JP3333902B2 - Transgenic animals, cells and cell lines obtained therefrom, and uses thereof - Google Patents

Abstract

The provision of cell lines from virtually any cell type of the animal body is greatly facilitated by transgenic non-human eukaryotic animals of the invention in which at least some cells have (i) a differentiation inhibiting sequence chromosomally incorporated under the control of a non-constitutive promotor and/or (ii) a differentiation inhibiting sequence which is itself conditionally active. Said genes are chromosomally incorporated under the control of a promotor such that expression of said sequence is normally held below an effective level, thus allowing normal cell development. However, cells taken from said animal may be prevented from completing differentiation to a non-dividing state in tissue culture by activating expression of said sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention provides a method for producing a chromosome, wherein the expression of the nucleic acid sequence is inhibited during normal animal growth, but the germ and / or somatic cells which can be activated in the isolated tissue culture are chromosomally integrated.
It relates to a non-human vertebrate, such as a mammal, obtained by "conditional" gene transfer. In addition, the present invention
It concerns such an isolated culture and its use in producing immortalized cell lines. Such cell lines also have many useful uses.

In the study of physiological functions at the cellular level, the availability of cell lines that enable biochemical experiments on homogeneous populations of cells from clones has greatly helped. Such cell lines have often been obtained from spontaneous or experimentally generated tumors. More recently, genetic engineering has made it possible to produce cell lines by inserting specific types of genetic information into the cell genome.

[0003] The various genetic information that enables the generation of cell lines share properties that prevent cells from differentiating into terminal, indivisible cells. Virology 127, 74-82 (1983) (Virolog
y 127,74-82 (1983)) and Petit et al.
l) describes the use of SV40 in the immortalization of rodent embryonic fibroblasts, and in many studies the expression of senescence, a phenotype usually seen after a limited number of fibroblast divisions The ability to rescue the entry of fibroblasts into the mold defines the differentiation inhibitory gene. With respect to the generation of cell lines, the entry of fibroblasts into such a senescent phenotype is sometimes referred to as a "crisis," and the ability to rescue cells from entering a crisis is a valuable tool for identifying genes in this family. A simple assay system. Since cells in which this particular type of genetic information is expressed can be established as a tissue culture system that grows in vitro for a virtually infinite period of time, the genes in this family are called established genes or immortalized genes (Land Others, Nature 1983, 304, 596-602, Lurie Nature 1983, 3
04, 602-606 (Land et al, Nature 1983, 304, 596-602,
Ruley Nature 1983, 304, 602-606)). These genes are also called maturation inhibiting genes because they prevent terminal differentiation.

[0004] Current analysis suggests that differentiation-inhibiting genes often belong to a family of oncogenes called nuclear oncogenes. These nuclear oncogenes are identified by several viral oncogenes (eg, SV) for which no cellular counterpart is known.
40 large T antigen, polyoma large T antigen, human papillomavirus E7 antigen) (Jat & Sharp 198)
6, Jay Virology 59, 746-750, Russell Zadegan, etc., 1982 Nature 300, 713-718, 1983 P.N.S.80, 4354-4358, Phelps et al., 1988 Cell 5
3, 539-547, and Petit et al. (Jat and Sharp 1)
986, J. Virology 59, 746-750, Rassoulzadegan et al, 19
82 Nature 300,713-718,1983 PNAS 80,4354-4358, Phe
lps et al, 1988 Cell 53, 539-547, and Petit et al su
pra)) and several genes for which cellular homologs are known (myc is the best example). In addition, there are genes in the family of cytoplasmic (or growth control) oncogenes that block differentiation in certain cell types. For example,
The src gene blocks the differentiation of progenitor cells. However, genes that appear to function in stimulating cell division rarely also have the ability to block the cell differentiation process.

[0005] In recent experiments, various methods of positioning immortalized oncogenes within cells have been used to enable the establishment of cell lines. For example, Molecular and Cellular Biology Vol 6, p. 1204-1217 (April 1986) (Molecu
lar and Cellular Biology Vol 6, p.1204-1217 (April
1986)), Jat et al. Reported that the mouse retrovirus shuttle vector system
It describes its use in the assembly of a recombinant retrovirus for infection of F111 cells. The resulting cell line expresses SV40 large T and cannot generate tumors,
It showed efficient microcolonization (in soft agar).
Furthermore, using these recombinant retroviruses, they
It has been shown that SV40 large T antigen alone can efficiently immortalize primary fibroblasts without crisis (Jat et al., Supra).

Although there are various transfection methods, retrovirus-mediated gene insertion is the most commonly used method for introducing immortalized genetic elements. Each of these methods has some disadvantages. First, there is currently no means to target a particular cell population. Second, poor efficiency of effective gene insertion (roughly, more than 1 per 10 ⁴ cells), therefore, the use of a large number of cells are required in order to establish some even regular cell lines . Third, effective integration of genetic elements requires induction of cell division in tissue culture. Fourth, cells must be extensively grown in culture before they can be used in experiments, and this growth is usually time-consuming under artificial conditions, such as applying very artificial selective pressure to the cell population. .

[0007] It would be very useful to have a method that could establish cell lines from various cell types more efficiently and reliably than is possible with current technology.

Further, all cells of the body can be divided into two different classes, precursor cells and end-stage cells. Progenitor cells (which are rogenitor ce
ll) and stem cells) are cells involved in the recruitment of specific cell populations in the body. They can be limited to the production of only one type of end-stage cell (eg, skeletal muscle precursors are thought to give rise only to skeletal muscle), and they can be bipotential (eg, granulocyte-macrophage primordial). Cells give rise to granulocytes and macrophages only) or they can give rise to many cell types (eg, hematopoietic stem cells give rise to all cells in the bloodstream, embryonic stem cells give rise to all cell types in the body) be able to). In contrast, end-stage cells are cells that have reached the end of their differentiation pathway and have lost the ability to generate multiple cell types, and more importantly, participate in recruiting damaged tissue populations. Have lost the ability to

[0009] Sufficient knowledge of specific progenitor cells that allows restoration of normal tissue function using primitive forms of precursor transplantation therapy that replaces both progenitor cells and differentiated end-stage cells There are a few examples that exist, all related to the hematopoietic system. This primitive form of precursor therapy removes hematopoietic stem cells from donor individuals (along with other cells) from the normal hematopoietic population (as is often the case as a result of radiotherapy for spread malignant tumors). It is a principle that supports widespread bone marrow transplantation, such as transplantation to a lost receptor. The infused hematopoietic stem cells colonize the patient's own bone marrow, and from this single stem cell population, megakaryocytes, lymphocytes, macrophages, eosinophils,
Produces a wide variety of other cells.

[0010] Although there are many present and future scientific and medical fields in which the ability to perform precursor replacement therapy is of great value, efforts in this area are currently contributing to the development of precursors that contribute to the normal development of most tissues of the body. It is hampered by a lack of current knowledge of the identity of the body group. Despite years of effort, including studies of these populations, there are only a limited number of examples where unique precursor populations can be identified and manipulated in tissue culture in a manner that can be later introduced into damaged tissue . Similarly, there are only a limited number of cases in which the identity of the molecular signal that divides or differentiates a precursor by a specific pathway is known. The acquisition of such tissues, which would be of great value in more effectively replenishing or repairing damaged tissues in the body's own progenitor population, is a suitable cellular assay for the purification of these important molecules. It is largely hampered by the lack of systems and the lack of suitable raw materials.

[0011] In WO 89/09816, McKay et al. Describe a general method of immortalizing cell lines in which growth promoting genes are introduced into vertebrate cells. The intent is that the function of this gene is controlled by external factors and that gene function can be regulated at will. Then, progenitor cells are grown with the activated gene, and the resulting cell population is
It is described that the condition is changed to a "non-permissive" condition to allow differentiation by inactivating the gene. At the 18th Annual Meeting of the Neuroscience Society in Toronto (November 1988) (see Abstract 14 (2) 1988 1130 of the Society of Neuroscience), Almazan et al, at the 18th Annual Meeting. of the
Society for Neuroscience, Toronto, (November 1988)
(See Soc.Neurosci.Abstr.14 (2) 1988 1130))
Immortalization of oligodendrocyte precursor cells using a temperature-sensitive oncogene carrier retrovirus.

However, while the use of the prior art to insert genes into progenitor cells has allowed the development of specific cell lines with the properties of a precursor, problems inherent in the generation of any cell line have been identified. Not only does it arise for the generation of cell lines, but the difficulties involved in establishing progenitor cell lines have the additional difficulty that precursors can represent only a fraction of the cells of a given tissue. The likelihood of successful introduction of genetic information into cells is correspondingly reduced.

[0013] So-called "transgenic" animals have also been known for several years, ie, animals that have incorporated into their animal genome a heterologous gene that can be expressed in a new chromosomal environment that alters the animal's characteristics in a designated manner.

A paper on transgenic animals was first published in the literature in 1982. In other words, Palmita et al. (Cell, 1982, 29: 7
01-710) (Palmiter et al. (Cell, 1982, 29: 701-71)
0)) was microinjected with a plasmid containing a mouse metallothionein-I promoter / regulatory site linked to the structural gene for herpesvirus thymidine kinase. They demonstrated the expression of hybrid genes in vivo and the regulation of genes in vivo by heavy metals. Gordon and Ruddle (Prog. C., Prog. Clin Violles, 1982, 85: 111-124)
Lin. Biol. Res., 1982, 85: 111-124) also demonstrated the inheritance of the injected DNA sequence. Palmiter and others (Nature, 1982: 611
−615) (Palmiter et al. (Nature, 1982: 611-615))
Showed that mouse gene transfer of growth hormone regulated by the metallothionein promoter grew abnormally large.

In 1983, McKnight et al. (Cell, 1983, 32: 335-34)
1), Lacey et al. (Sell, 1983, 34: 343-358), Palmiter et al. (Science, 1983, 22: 809-814), Brinster et al. (Nature, 306: 332-336), and Gordon (J. Exp)・ Elephant, 1983, 228: 313-324)
(McKnight et al. (Cell, 1983, 32: 335-341), Lacy et.
al (Cell, 1983, 34: 343-358), Palmiter et al. (Scie
nce, 1983, 22: 809-814), Brinster et al (Nature, 30
6: 332-336), and Gordon (J. Exp.Zool., 1983, 228: 313).
-324)) reported on the specificity of transgenic animals without touching cell growth in tissue culture.

[0016] In 1984, the first paper was published that pointed out that cancer cell expression prevents normal development. Brinster et al. (Cell, 1984, 37: 367-379) (Brinster et al. (Cell, 198).
4,37: 367-379) showed that mice expressing the SV40 large T antigen under the control of the metallothionein promoter had tumors of the choroid plexus. Tumors develop later in life, indicating that not only transgenes were involved in tumor formation. Although cell lines were obtained from tumor tissue, no attempt was made to obtain cell lines from pre-tumor or other tissues. A little later,
Stuart et al. (Cell, 1984, 38: 627-637) (Stewart
et al. (Cell, 1984, 38: 627-637) report that c.c. under the control of the hormone-induced mouse mammary tumor virus promoter.
We reported that mammary carcinoma developed in mice expressing -myc. No cell line was described. For the potential use of cells from transgenic mice in culture, see Ritches et al. (Nature, 1984, 312: 517-520).
(Ritchies et al. (Nature, 1984, 312: 517-520)), which makes hybridomas by fusing spleen cells derived from a mouse transgene for the immunoglobulin kappa gene with a normal hybridoma fusion partner. It shows that it is possible.

In US Pat. No. 4,736,866, Leder and Stewart describes a non-human transgenic mammal whose germ and somatic cells contain an active oncogene sequence. Such animals are particularly useful models for testing anti-cancer drugs because of their strong tendency to develop neoplasms and the low levels of drug dosage used in the test. However, the animals show a pronounced tendency to develop abnormally, which renders them substantially useless as models for studying normal cell development or as a source of therapeutic biomaterials.

[0018] Palmita and others (Nature, 1985, 316: 457-460)
(Palmiter et al. (Nature, 1985, 316: 457-460))
Continues the previous analysis of the effects of SV40 large T antigen in transgenic mice and shows that the development of choroid plexus tumors requires the presence of the SV40 enhancer site and
The SV40 construct (structure) was shown to give rise to heterogeneous tumors. Cell lines were isolated from several mouse hepatomas, indicating that these cells express T antigen in their nuclei. The authors describe producing mice with a ts58 temperature-sensitive derivative of SV40, but state that this construct is not temperature-sensitive. The widespread use of the T antigen in the production of transgenic mice and the use of ts58 (=
(tsA58) Despite the widespread use of the mutant, other previous studies, including those of Palmiter and his colleagues, did not return to this mutant.

The overall goals of the gene transfer studies reported so far are:
It can be said that the introduction of genetic information that interferes with the normal development of roughly specific tissues into the germ line. In stark contrast, as will become apparent, the present invention provides that the present invention does not interfere with normal development, cells are harvested, and in particular, activation of the transgene in tissue culture is potentially possible. It concerns the production of transgenic animals, which facilitates the study of cells from all tissues of the body as a gender.

Therefore, for example, in other research institutions, even if a gene construct using a regulatable promoter (H-2K ^b ) to regulate cancer cell expression has been produced, it is not The possibility that the use of such a promoter would enable conditional oncogene expression in vitro was not previously recognized. The most detailed example is described in Morero et al. (Cancer Cell Research, 1989, 4: 111-125) (Mo
rello et al. (Oncogene Research, 1989, 4: 111-12
5) The H-2K ^b promoter described by c)
A study linked to the myc gene, which created several transgenic strains carrying the fusion gene, to determine if constitutive c-myc expression was found in all tissues, and to confirm the biological effects of enhanced myc constitutively expressed in these transgenic animals, 5'H-2K ^b promoter sequence human c-myc
It is linked to the proto-oncogene. The author obtained 33 mice leading to the establishment of five transgenic lines. The authors report that expression of the H-2 / myc construct was found in most organs analyzed, with maximal expression in lymphoid organs and minimal expression in brain and liver. H-2K
The level of / myc expression corresponded to H-2K expression. Morello et al. Reported that 20 H-4K / mcy mice
They also reported no disease for a period of months, which concludes that immortalization with this construct requires a second genetic event.

In previous gene transfer studies, Eflat and Hanahan (Mol cell viol, 1987, 7: 192-198) (Efrat
and Hanahan (Mol. Cell Biol., 1987, 7: 192-198))
Investigated the cell-specific activity of the reverse promoter element in two lines of transgenic mice using the promoter to target T antigen expression to pancreatic β-islet cells.
Gene expression was examined in tumor cells. Also, Eflat et al. (Plock Natl Acad Sai U.S.A., 1988, 85: 9037-9041 (Efrat et al. (Proc. Natl. A.)
cad. Sci. USA, 1988, 85: 9037-9041) describes the action of three pancreatic β-cell lines established from islet cell tumors obtained from transgenic mice carrying the hybrid insulin promoter SV40T antigen gene. Was examined. All primary β
Beta tumor cells from cell tumors maintained the characteristics of beta cells over 50 passages in culture. The authors conclude that "targeted expression of oncogenes with cell-specific regulatory elements can be used both to immortalize rare cell types and to select for the maintenance of their differentiated phenotype." I have. In the present invention, an important contribution to this scientific field is that "targeted expression" is unnecessary. As will become apparent below, the transgenic animal of the present invention can be a cell-type storage that can be optionally selected and employed for immortalization as needed.

[0022] In yet another gene transfer study, Biebrich et al.
Cell Viol, 1987, 7: 4003-4009) (Bieberich et.
al. (Mol. Cell Biol., 1987, 7: 4003-4009) also used transgenic mice to study class I antigen function, and skin grafts from transgenic mice were rapidly transferred to background strain mice. It was found that they were rejected and that the class 1 transgene could be induced by interferon treatment and suppressed by human adenovirus 12 transformation.

In 1987, Choi et al. (Jay Byrol, 198
7, 61: 3013-3019) (Choi et al. (J. Virol., 1987, 61:
3013-3019)) examined the expression of the simian virus 40 early region gene, which is transcriptionally regulated by the mouse mammary tumor virus long terminal repeat. Cells cultured from the transgenic animals showed glucocorticoid-inducible chimeric gene expression. Many, if not all, tissues expressing the Simian Virus 40 sequence exhibit premalignant characteristics,
Has developed into a tumor.

In 1988, Paul et al. (Klin Bohenstra, 198
8, 66, Sapple 11: 134-139; Ex-Sell-Less, 17
5, 354-365) (Paul et al. (Klin Wochenstr., 1988, 6).
6, Suppl. 11: 134-139; Exp.Cell Res., 175, 354-365))
Created a permanently growing hepatocyte line by growing hepatocytes from mice expressing the SV40 viral sequence induced by the mouse metallothionein enhancer sequence. Most hepatocytes show an immortalized phenotype in culture and grow further and become more and more transformed in culture. The original cells were non-malignant, but clearly different from normal cells in that they did not require the addition of epidermal growth factor to chemically defined media to promote cell division. In vivo, mice developed hepatocellular carcinoma.

[0025] McKay et al. (Kidney Int, 1988, 33: 677-68)
4) (MacKay et al. (Kidney Int., 1988, 33: 677-68)
4)) established a permanent cell line of cloned glomerular epithelial, mesothelial and endothelial cells from the mouse transgene system for Simian virus 40. These mice appear normal at birth, but after 3-4 months a variable percentage of sclerosis affects the glomeruli. Cells obtained from these mice maintained the characteristics of their normal counterparts despite a transformed phenotype.

Landon et al. (Oncogenes Les, 1988, 3: 271-27)
9) (Langdon et al. (Oncogene Res., 1988, 3: 271-27)
9)) performed an in vitro study of the E mu-myc gene-introduced B-lymphoid cells to examine their progression to lymphoma characteristics. As a result, the cells initially require a bone marrow feeder layer, after which the culture degrades to a monoclonal or oligoclonal composition, and achieves growth autonomy only at a later time,
Thus, the importance of a number of events in establishing growth autonomy was demonstrated.

In the above WO 89/09816, the conditional immortalized cells described therein can be introduced into an animal to produce a transgenic animal in which the growth promoting gene present in said cells is inactive at normal body temperature. Sex is suggested. However, the studies described do not address this suggestion, and there is no mention of how such techniques could be successfully applied. There is no disclosure of a promotion system or conditional oncogene that produces only low levels of expression as used in the present invention (see below), cells, differentiated or progenitor cells (which can be immortalized later in culture) There is no disclosure of the use of stable transgenic animals as a source.

It will be apparent that it would be highly desirable to provide a method for efficiently obtaining cell lines from potentially any tissue of the body. This is a goal that has heretofore been far from realizable, apart from the specific problems mentioned above, and despite the great interest in transgenic animals.

In view of the above-mentioned conventional problems and goals, the present invention relates to a transgenic, non-human, eukaryotic animal having a germ cell and / or somatic cell in which a sequence is integrated into a chromosome. The sequence encodes a differentiation-inhibitory product that becomes conditionally active, and the sequence is under the control of an activatable promoter, wherein in vivo, the activatable promoter is The animal is controlled so that the expression of the sequence is inhibited, and the animal is subjected to the conditions under which the conditionally active differentiation-inhibiting product is inactivated, so that the animal is expressed even if the differentiation-inhibiting product is expressed. Have a functional level low enough to allow normal development of the cells in culture, and in culture, under permissive conditions, i.e., the activatable promoter is In addition to performing control such that expression of the sequence is induced, and given the conditions under which the conditionally active differentiation-inhibiting product becomes active, the differentiation of cells removed from the animal is completed. Characterized by having a sufficient level of functional expression to prevent,
Provided are transgenic, non-human, eukaryotic animals having germ cells and / or somatic cells whose sequences have been chromosomally integrated. As described below, in the animal of the present invention, the sequence preferably encodes TAgts. It is preferable that the promoter is an HLA class I promoter, more preferably said promoter is an HLA H-2K ^b promoter. Further, it is preferred that the promoter has a plurality of operably linked genetic regulatory elements, and that the animal is a mouse or a rat.

[0030] In one aspect, the present invention provides a gene having a germ cell and / or a somatic cell in which a sequence encoding a differentiation inhibitory product that is conditionally activated under the control of an activatable promoter is chromosomally integrated. Introduced, non-human, eukaryotic animals, wherein control of the activatable promoter usually involves controlling the expression of the sequence to be inhibited so as to enable normal cell development. However, if the progenitor cells removed from the animal are placed in tissue culture,
By providing a permissive state to the promoter and activating the expression of the sequence, control is performed so as to prevent the differentiation of the progenitor cells removed from the animal in tissue culture from being completed. It is intended to provide a transgenic non-human or eukaryotic animal having a germ cell and / or a somatic cell into which a sequence encoding a conditionally active differentiation inhibitory product has been integrated into a chromosome. In connection with these "conditional" transgenic animals, an "activatable promoter" as used herein is intended to (a) produce, under its control, a much higher level of expression of a sequence than if not induced. Induced and (b) means a promoter system that, when not induced, does not permit detectable expression or only permits expression at levels that do not interfere with normal cell development. It will be appreciated that a promoter system that is "leaky" or allows for low expression is acceptable as long as the level of expression does not interfere with normal developmental patterns. As will become clear later, there are various methods for inducing the expression level in animals to be low, but inducing an increase as needed. Thus, "conditional" can be achieved using a number of means, as in the particular study described herein using a dual conditional form involving conditional oncogenes. For example, multi-conditional alternatively relies on the use of a promoter system employing two or more regulatory genetic elements, all of which are required to achieve the high level expression described in (a) above. can do.

In another aspect, the invention relates to a transgenic, non-human animal comprising a cell into which a sequence encoding a differentiation-inhibiting product that is conditionally active under the control of an activatable promoter is integrated into a chromosome. Wherein expression of the sequence is inhibited unless activation of the activatable promoter.

The fully differentiated cell type can, if desired, be effectively placed in tissue culture from such animals, and furthermore, the present invention provides a method for obtaining progenitor cells from normal tissue. Which are performed in a manner that greatly enhances the likelihood of understanding not only the identity of these cells, but also the biological principles that regulate their development.

The above-mentioned invention can produce a transgenic animal in which tissue development and differentiation can be performed normally in vivo by achieving a low expression level in vivo without induction. Provides a treasure trove of materials.

In general, the invention described above provides for a conditionally active differentiation that is inactive in most or all tissues of normal animals, and in particular is configured to have a minimal effect on normal development of the animals. Gene transfer, non-human, with germ and / or somatic cells containing a DNA sequence encoding an inhibitory product,
For eukaryotes. Activation of a gene construct that can prevent terminal differentiation is achieved preferentially through manipulation of the dissected tissue in vitro, although the construct can be activated in vivo.

Thus, in another aspect, the invention relates to a gene transfer comprising a germ cell and / or a somatic cell having a chromosomal integration of a sequence encoding a differentiation-inhibitory product that is conditionally active under the control of a promoter. A non-human, eukaryotic animal, wherein the control of a promoter usually means controlling the expression of the sequence to be maintained below an effective level so as to enable normal cell development. However, if the progenitor cells removed from the animal are placed in tissue culture,
By activating expression of the sequence, it becomes conditionally active that controls such that progenitor cells removed from the animal can be prevented from completing differentiation in tissue culture. It is intended to provide a transgenic non-human or eukaryotic animal having a germ cell and / or a somatic cell into which a sequence encoding a differentiation inhibitory product has been integrated into a chromosome.

In animals of the type described above, the conditionally active differentiation inhibitory product may be TAgts and / or the promoter may be Tgts.
A "weak" non-inducible promoter, such as the K promoter, may be used. Molecular manipulation of any of a number of promoters (eg, Levin & Manly, Cell, 1989, 59: 405-
408, Able and Maniatis, Nature, 1
989, 241: 24-25, and Michel and Cian, Science, 1989, 245: 371-378 (Levin and Manl
ey, Cell, 1989, 59: 405-408, Abel and Maniatis, Nature,
1989, 341: 24-25, and Mitchell and Tjian, Science, 198.
9,245: 371-378), it will be appreciated by those skilled in the art that it is possible to generate weak, non-inducible promoters suitable for use in the above invention.

In another aspect, the invention includes a cell isolated from the animal, or obtained from a cell so isolated, wherein the sequence capable of activating expression has been chromosomally integrated.

The invention also includes a cell line obtained from such a cell and immortalized by activating expression of said sequence.

[0039] In yet another aspect, the invention provides a method for preparing a cell from the above cells by allowing differentiation without activating the expression of the sequence, or from the above cell line by inactivating the expression of the sequence. Alternatively, it includes a differentiated cell which activates the expression of the sequence but is capable of inducing differentiation of the cell by exposure to an external factor, and which is obtained from the cell so exposed.

The present invention also provides a method for producing a transgenic, non-human or eukaryotic animal, wherein the animal has a construct having a sequence encoding a differentiation-inhibiting product that is conditionally active, and The sequence is under the control of an activatable promoter, wherein the differentiation-inhibiting product controls, in vivo, such that the activatable promoter inhibits expression of the sequence, and The differentiation inhibitory product, even when expressed by such conditions that the differentiation inhibitory product that becomes active becomes inactive, has a sufficiently low function to allow normal development of the cells in the animal. The differentiation-inhibiting product is only allowed to stand in culture during permissive conditions, that is, when the activatable promoter performs control such that the expression of the sequence is induced. In particular, given the conditions under which the conditionally active differentiation-inhibiting product becomes active, it has expression of a functional level sufficient to prevent the completion of differentiation of cells removed from the animal. A method for producing a gene, introducing a non-human or eukaryotic animal, comprising performing chromosomal integration of the construct into at least a part of cells of the animal.

Although the technique used to achieve gene transfer is not critical to the inventive concept, microinjection at the embryonic stage using well known procedures is usually the preferred means. Microinjection can be used at any stage of development, from the single cell stage to later embryonic stages.

In the above method of the present invention, it is preferable that the sequence encodes TAgts. Preferably said promoter is an HLA class I promoter, more preferably said promoter is an HLA H-2K ^b promoter. It is also preferred that the promoter has a plurality of operably linked genetic regulatory elements, and that the animal is a mouse or a rat.

However, transgenic animals can be produced by various methods all within the scope of the invention.
The genetic construct can be inserted into embryonic stem cells, and these engineered stem cells can be injected into fertilized zygotes at a stage where a small number of cells are present. Embryonic stem cells can be successfully integrated into the zygote, and these genetically engineered cells can differentiate to form many or all cell types present in the body. In some animals, such cells also contribute to the germ line, thus providing a means to make fully transgenic animals as progeny of the early chimeras. It has also been suggested that sperm itself can be used as a vector to generate transgenic animals (although this claim is now debatable) and may be incorporated as a result of events during the pre- conception stage. Should not be ignored. If the sequence encoding the differentiation inhibitory product is expressed in at least some cells of the transgenic animal under the control of an activatable promoter, and thus the sequence is regulatable,
The manner of achieving chromosome integration and the timing (during animal development) according to the present invention are not critical.

A central feature of the present invention is the intention to avoid the expression levels of genetically introduced genetic information that would interfere with normal development. This feature distinguishes the present invention from all other previously reported gene transfer experiments. This need of the present invention is primarily achieved by utilizing a promoter sequence that is to be specifically activated to allow significant expression of the differentiation inhibitory gene. In the non-limiting examples described below, the effects of oncogenes are further restricted by using oncogene sequences encoding differentiation-inhibitory products that are only conditionally active.
Thus, the invention employs the principle of multiple conditions. This is accomplished, for example, by the use of multiple genetic regulatory elements that when used together provide the above-described activatable promoter system, or that, when used together, This can be achieved by the use of one or more genetic regulatory elements to achieve the desired effect of providing the system and a conditional oncogene-like sequence.

The differentiation inhibitory product encoded by the sequence according to the present invention is capable of inhibiting the differentiation of progenitor cells. Such sequences or genes include oncogenes that encode proteins localized in the cell nucleus. Some of the products encoded by these nuclear oncogenes immortalize cells (and thus give them the ability to grow indefinitely (indefinitely) without entering a terminally differentiated state), and progenitor cells undergo non-dividing, terminal stages. Has the ability to inhibit differentiation into cells. Some of these genes are not only of viral origin,
At present, there is no known mammalian counterpart in the normal genome. For example, the SV40 large T antigen, the human papillomavirus E7 protein, and the polyoma large T antigen all have the ability to interact with normal cellular proteins in a manner that appears to be involved in the action of the viral proteins in neoplastic transformation. Although shown to be the case, the normal cellular counterparts of these viral proteins are not currently known (White P et al., 1988 Nature 388, 124-129, Decaprio J.A. et al., 1988 Cell 54, 275-283, and Dyson
N et al., 1989 Science 143, 934-937 (Whyte P. et a
l, 1988 Nature 388,124-129, De Caprio JAet al, 198
8 Cell 54,275-283, and Dyson N. et al, 1989 Science
243, 934-937)). Some normal cellular proteins such as c-myc localized in the nucleus have the ability to immortalize cells and inhibit progenitor cell differentiation (Land et al., Dot-GP et al., 1985 Nature, supra). 318, 472-475,
And Dmitrovsky Y et al., 1986 Nature 31
1, 748-750 (Land et al supra, Dotto GP et al, 1985)
Nature 318,472-475, and Dmitrovsky E. et al, 1986 N
ature 311,748-750)). In addition, a small number of oncogene products, such as src, that may be associated with growth regulation are capable of immortalizing certain precursor populations and inhibiting their differentiation.

In general, it can be said that there are currently five different gene groups that can function as differentiation-inhibiting sequences in the present invention, ie, the sequences encoding differentiation-inhibiting products. Some of the known genes in each category are listed in the table below. The first gene category belongs to the nuclear cancer gene family and includes genes such as SV40 large T and genes such as myc and myb. The second category of genes is that which can be converted from a suppressor gene to an immortalized gene by mutation. Currently the only known representative of this category is p
^{At 53} , it appears to interact in a previously unknown manner with proteins of the family that are also involved in modulating retinoblastoma gene product activity. The third gene category, which is generally thought to be involved in controlling cell proliferation, appears to be capable of inhibiting the differentiation of certain cell types. The fourth gene category is typically a secreted molecule called differentiation inhibitory activity, and seems to have an action of inhibiting differentiation of embryonic stem cells via cell surface receptors. Finally, it was recently discovered that cooperative interactions between mitogens can also inhibit progenitor cell differentiation. In a study on rat optic nerve oligodendroglial type 2 stellate progenitor cells, when these progenitors were stimulated simultaneously with platelet-derived growth factor and basic fibroblast growth factor, they seemed to have mutational activity of nuclear oncogenes. In tissue culture without transformation, it was found that the differentiation of precursors into oligodendrocytes was completely inhibited, allowing indefinite (infinite) growth of 0-2A progenitors (Bogler et al., 1990, Plock Natl Akad Sai USA, 87: 6368-6372 (Bogler et al., 1990, Pro
c. Natl. Acad. Sci. USA. 87: 6368-6372)). The last two sets of results indicate that it is also possible to use soluble factors controlled by an inducible activatable promoter to inhibit cell differentiation. In such a situation, the "differentiation inhibiting sequence" used in the present invention is a gene sequence encoding the above factor.

[0047]

Table 1 Nuclear oncogene SV40 large T polyoma large T adenovirus EIA HPV E7 and E6 mycer A myb dominant mutation-modifying tumor suppressor gene Mutants of p53 Growth-regulating genes that inhibit differentiation V-src Gene producing differentiation inhibitor Differentiation inhibitory activity Gene sequence encoding a combination of multiple growth factors acting to inhibit differentiation Platelet-derived growth factor + basic fibroblast growth factor

A list of nuclear cancer genes that are very often genes with the ability to inhibit differentiation has recently been compiled by Hunter (Cell, 1990, 64: 249-270 (Cell, 1990, 6).
4: 249-270)).

[0049] The differentiation-inhibiting sequence used in the present invention is contained in the genome in such a manner that the potential of the immortalizing gene to function is limited in vivo. For example, a thermolabile form of the SV40 large T antigen (TAg) (Tegtmeier, 1975 J Virology 1)
5, 613-618 (Tegtmeyer, 1975 J. Virology 15,613-61
8)) Can be used, it is normal body temperature of mouse (39.5 ℃)
Rapidly degraded and thus inactivated. In an embodiment of the invention, this temperature sensitive TAg (TAgts) is under the control of a promoter that normally controls the expression of the class I antigen of the major histocompatibility complex (Kimura et al., 1986 cells 44, 261-27).
2, and Baldwin Jr. AS, etc., 1
987 mol cell viol, 7, 305-313 (Kimura et a
l.1986 Cell 44,261-272, and Baldwin Jr.ASet al, 1
987 Mol. Cell Biol. 7, 305-313)). This promoter can be activated by exposure to gamma interferon, but is usually only active at low levels in most tissues of healthy animal bodies.

The basis for the use of a controllable activatable promoter is to inhibit the expression of the intended gene, unless expression is desired. The activatable promoter that can be used in the present invention can be controlled by changing conditions. Under certain conditions (non-permissive), promoter action is inhibited, resulting in normal cell development. When the conditions are changed tolerable (eg, by exposure to gamma interferon in the above example), a significant degree of expression occurs. If the conditional oncogene is allowed to be constantly expressed in all tissues, then the reversion of the oncogene itself is prone to abnormal development, and if a sufficiently high level of the gene product is expressed, That the low level activity of the conditional oncogene can be effective (as described in Example 1 below) and that the expression of a functional level of the wild-type differentiation inhibitory sequence in vivo results in tissue transformation ( For example, as is known in other gene transfer models as disclosed in U.S. Pat.
It is imperative to avoid significant expression.

A reversion caused by a random mutation is
It occurs at a frequency of 1 in 10 ⁶ cells. Animal body is 10 ¹²
This means that many cells in the body will express the conditional gene backmutation, as it contains much more cells. In addition, experiments in tissue culture have shown that reversions associated with other cell regulatory pathways can occur as frequently as 1 in 10 ⁴ . Thus, it is likely that each body tissue has, for example, a large number of cells expressing a functional oncogene capable of inhibiting the normal pathway of differentiation. Such a situation is contrary to normal development. In fact, one of the inventors (P. Jat) and his colleagues,
An undisclosed attempt was made to create transgenic mice in which Agts was placed under the control of the beta-actin promoter and TAgts was constitutively expressed at significantly higher levels in each cell of the body. Such a genetic construct does not appear to be suitable for the survival of the engineered embryo. Therefore, in order to avoid the problem of reversion, it is essential to use a promoter that reduces the expression of TAgts to below the effective level.

Other possible promoter systems for use in the present invention include those based on lactose (lac) -inducible operons isolated from bacteria. Rack-inducible systems are based on having a binding site for a particular protein located between the transcription promoter and the transcription start site. Usually, the repressor associates with the operator and causes steric hindrance of transcription. The presence of inducers in the cells complexes the repressor and prevents it from binding to the operator, thereby allowing transcription. The specificity inducer used is the non-metabolizable, non-naturally occurring allolactose analog IPTG. Lactose can also activate this inducer, but lactose is rapidly metabolized and presently has low levels in almost all tissues. The only bodily fluid that contains high levels of lactose is milk, which increases the likelihood that this construct will be induced in lactating female milk producing cells. Unlike class I antigens, which are constitutively and inducibly expressed by a large number of cells (and consequently have higher expression levels than before induction), the rack repressor is a class of differentiation-inhibiting genes used in the transgenic animals of the present case. More reliable control of expression can be performed. Of course, it is known that the rack system works with mammalian cells.

The rack-inducible promoter is a known example of a bacterial promoter that can be activated by substances that are unlikely to induce induction in normal warm-blooded animals, but the discovery of other similar bacterial promoter systems is contemplated. Can be In addition, the use of mutant forms of the bacterial promoter may be possible, as exemplified by wrap mutations in the rack repressor.
Such a promoter is used in the method of the invention.

Yet another example of an inducible promoter is the metallothionein promoter, which is activated by heavy metals such as zinc, cadmium and the like. This promoter can also be used in the method of the present invention.

The glucocorticoid-regulated MMTV promoter is an inducible promoter used in gene transfer experiments. However, this promoter may endogenous glucocorticoid production may activate the expression of the differentiation inhibitory construct.

Apart from applicability throughout the animal kingdom (see below), there are at least two advantages of the class I promoter system. First, in tissues with low levels of endogenous class I antigen expression, the activity level of this promoter is:
For example, it is too low to allow production of the TAgtsA58 antigen sufficient to interfere with normal development. However, the addition of an inducer (eg, gamma interferon) can super-induce the activity of this promoter and bring the level of the TAgtsA58 antigen to a level where full activity of the differentiation inhibitory gene is seen. Furthermore, the use of this promoter can induce the expression of TAgtsA58, for example, in tissues that normally do not express class I antigens (eg, the central nervous system), since all cells have a functional interferon receptor. Because.

A great advantage of the present invention is that its general concept is applicable to all species of warm-blooded animals. For example, in the above example, the class I promoter usually causes high level expression of the major histocompatibility complex gene when cells are exposed to gamma interferon (Walach D. et al., Nature 299, Q33-836 (Wallach D. et al, Natur
e 299, Q33-836)). This gene activation pathway is human,
It is already well known that it occurs in rodents, rats and mice, and therefore is thought to occur in all warm-blooded animals. Furthermore, as already mentioned, bacterial lac promoters are known to function in mammalian cells.

The animals of the present invention can potentially be used as raw materials for the growth, identification, purification and detailed analysis of progenitor cells from potentially all tissues of the body (Moston G et al., Hematopoietic Growth Factors, A Review Cancer Research 1988, 48, 5624-5637 (Morston G. et al, Hemopo
ietic Growth Factors, A. Review Cancer Research 198
8,48,5624-5637)). The progenitor cells can be grown indefinitely (indefinitely) by inserting the cut tissue into tissue culture under conditions that activate the immortalization / differentiation inhibiting gene. Furthermore, cells that are not normally considered progenitor cells (such as fibroblasts) can also be immortalized by experimental manipulations that form part of the present invention.
As indicated earlier, the animals of this invention are not suitable for testing for carcinogens or for those that are believed to provide protection against neoplastic development in US Pat. No. 4,736,866.
Different from animals. Generally, the tissues of the animals of the present invention undergo normal development until placed in tissue culture. In the present study, normal development was seen except in the thymus (indicating increased delayed hyperplasia of the whole organ), and even within the thymus, the function of the genetic construct to prevent terminal differentiation was conditional. is there. Cells placed in an abnormally developed state (as described in U.S. Pat. No. 4,736,866) do not exhibit the properties of normal cells and such cells tend to undergo additional mutations that activate oncogenes. There is evidence that there is. Therefore, cells collected from the tissue of an animal that expresses an activated oncogene in vivo may not be reliable as a suitable model for studying normal cells, especially for studying normal progenitor cells. In contrast, cells isolated from the animal tissues described in the present invention are considered to have undergone normal development, and once grown in vitro, once the cells express the immortalizing protein, the normal It is thought that it approaches as much as possible the corresponding thing.

Accordingly, in another aspect of the present invention, there is provided a method for producing an immortalized cell, comprising separating a precursor or a differentiated cell into which the sequence has been chromosomally integrated, from the animal of the present invention, Is placed in tissue culture conditions in which expression of said sequence is activated.

In particular, the present invention relates to a method for establishing cells in culture, comprising the steps of removing the cells from the animal of the present invention, and permitting the removed cells in culture under permissive conditions, ie integrating the chromosome into said cells. The expression of the sequence encoding the conditionally active differentiation inhibitory product is induced by the control of a non-constitutive promoter, and the conditionally active differentiation inhibitory product encoded by the differentiation inhibitory sequence is activated. Wherein said permissive conditions lead to the expression of a sufficient functional level of said differentiation-inhibiting product to prevent the differentiation of said cells from being completed. A method for establishing a strain is provided.

The cells removed in this method include pancreatic cells,
Insulin-producing cell precursors, glial cells, glial cell precursors,
Cells selected from the group consisting of muscle cells and muscle cell precursors are preferred. This method further comprises a step of inducing differentiation by giving an external factor to the culture, which also forms a part of the present invention. Isolated cells obtained by such a method, in particular, as described below, also obtained by such a method for the treatment of the human or animal body also form part of the invention. The present invention also provides a method for producing an expression product of a cell obtained by such a method, comprising culturing the cell under conditions suitable for producing the expression product, as described above. Also provided is a method for producing an expression product of a cell obtained by the method.

[0062] Prevention of levels of oncogene expression that disrupts normal development can also be achieved theoretically by manipulating the promoter system to create a more subtle regulatory mechanism than exists in a single genetic control element. . For example, a theoretically useful promoter system is described by Reed et al., Plock Natl Acad Sai U.S.A., 1989, 86: 840-84.
4 (Reid et al., Proc. Natl. Acad. Sci., USA., 1989, 86:84
0-844), wherein the thymidine kinase promoter (constitutively expressed at low levels)
Is placed downstream of a short (18 nucleotide) sequence that is sufficient to confer interferon inducibility to the TK promoter. This promoter construct can theoretically be used to drive tsA58 expression and reduce the basal level of expression while retaining interferon inducibility.

Extending the principle of dual conditionality provided in this document, SV40
The expression of the heat-labile TAgtsA58 mutant can be controlled by the promoter element of the class I antigen gene. This can be extended by the use of other temperature-sensitive genes capable of inhibiting normal differentiation.
In addition, it is possible to create chimeric oncogenes that are conditional to contain hormone receptor sequences essential for oncogene function. Examples of such proteins are Picard et al. (Cell, 1988, 54: 1073-1080) (Picard et al. (Ce
Illers et al. (Nature, 1989, 340: 66-68) and Eilers et al.
1989, 340: 66-68)), which make an adenovirus ElA protein with the rat glucocorticoid receptor hormone binding domain and a myc protein with the human estrogen receptor binding domain, respectively. Was. In both cases, the effect of the chimeric protein on host cell function depends on the binding of the appropriate hormone to the chimeric protein.

In a particular embodiment of the invention, the differentiation inhibitory sequence or gene used encodes a temperature-sensitive large T antigen from simian virus SV40. Utilization of this gene provides a second level of control over gene activity during normal development, as the protein encoded by this mutant gene is rapidly degraded at temperatures close to normal body temperature in mice. However, as described elsewhere herein, utilizing a temperature-sensitive gene in combination with a constitutive (ie, non-regulatable) promoter, when the promoter is as strong as, for example, the beta actin promoter, There is sufficient evidence that it cannot be expected to be suitable for normal development. Thus, other differentiation-inhibiting genes can be used as long as they can be regulated by an activatable promoter that is inactive in most or all tissues of normally developing bodies.

The primary cells used in the studies described below to illustrate the invention were skin, thymus, pancreas, central nervous system,
Cells from colonic crypts, endothelium, skeletal muscle, and intestinal glial cells. However, it is to be understood that the method of the present invention can be used to immortalize virtually any type of cell obtained from the body of a suitable transgenic animal.

Since the particular cells selected to illustrate the description herein are from tissues with extensive cell characterization, the cell lines obtained with the methods of the invention are expected to be normal cells It can be confirmed that the desired properties are exhibited. Thus, these cell lines are a direct demonstration that the method can be used to produce a wide variety of cell types of continuous cell lines (passage cell lines) from a wide variety of tissues. Furthermore, some of the studies described herein directly demonstrate the utility of animals in producing cultures where new cell types are amenable to study.

To confirm that the general principles embodied in this invention are correct, skin was used as the primary source of fibroblasts. Skin cells were placed in tissue culture at a temperature that allowed the expression of the immortalizing effects of TAgts under conditions that activated the promoter used. These cells do not activate the promoter, are non-permissive for the action of TAgts,
They were grown for various lengths of time before switching to both conditions. As shown in FIG. 2 for the different transgenic mice, removal of the promoter activating compound (here mouse gamma interferon) was associated with reduced cell growth rate. Growth of cells from most animals shows that cells are free of gamma interferon and 39.5
C. This ability to continue to grow at a low rate, but not in the absence of gamma interferon, was not completely suppressed unless grown at C.degree.
This is likely due to low constitutive expression levels of the antigen (Israel Ai et al., Nature 322, 743-746 (Isr
ael A. et al, Nature 322, 743-746)). Indeed, it is noteworthy that even the low constitutive expression level of this promoter found in normal fibroblasts was not associated with the development of any overt hyperplasia of the skin. FIG.
Continued expression of the higher copy number of the gene of interest, even when grown at 39.5 ° C. in conditions without interferon (cultures from animals 11 and 36 in the studies described below). It can be linked to slow growth. This slow growth is similar to that seen when gamma-interferon-depleted cells are grown at 33 ° C, presumably before inactivation by degradation at this non-permissive temperature (multiple gene copies). Due to the breakthrough of the activity of the large amount of T antigen produced.

The removal of gamma interferon and growth at 39.5 ° C., when the activity of differentiation-inhibiting genes and differentiation-inhibiting products is terminated, indicates that fibroblasts are in a non-dividing state by TAgt.
Similar to that observed in previous studies on the effects of immortalization of fibroblasts by s. In this earlier study, TAgts expression was regulated by a constitutively active viral LTR,
Using retrovirus-mediated gene insertion to generate a cell line that expresses only a single copy of the TAgts gene), cells grown at 33 ° C grow indefinitely (infinitely) in tissue culture. did it. On the other hand, when the cells were switched to 39.5 ° C., they rapidly lost their ability to undergo further cell division (Jat and Sharp, 1989, Mol. Cell Viol 9, 167-1681 (Jat and Sharp 1989, Mol. Cell,
Biol. 9,1672-1681)).

It is of particular interest that the conditionally immortalized fibroblasts become non-dividing, as to how this non-dividing state reflects the normal differentiation pathway of fibroblasts. Normal fibroblasts undergo a limited number of divisions before reaching a senescent state that exhibits normal metabolic function except that they are refractory to further cell division (Hyfrick El et al., 1961, Ex Cell Les, 25, 285, Todaro et al., 1963 Jay Cell Viol 17, 299-313 (Hayflick L. et al, 1961 Exp. Cell R
es. 25,285, Todaro et al, 1963 J. Cell Biol. 17, 299-31.
3)). The phenotype expressed by conditionally immortalized fibroblasts when switched from permissive to non-permissive conditions resembles indistinguishable from normal aging. Thus, conditionally immortalized cells, when grown under non-permissive conditions, can undergo a differentiation event of their normal counterpart.

[0070] Two of the cell lines developed from the thymus are defined as epithelial cell lines by the expression of cytokeratin, to exemplify the invention. The origin of epithelial cell lines is particularly interesting in view of their importance in human cancer, and the origin of thymic epithelial cell lines is even more interesting in view of their importance in the development of thymic T lymphocyte populations. . Cell lines were obtained from the thymus as many of the transgenic mice produced are prone to thymic hyperplasia. All cell lines derived from this tissue behave conditionally in tissue culture at 39.5 ° C.
Caused growth inhibition when grown without interferon. The condition of these cell lines in vitro is such that, even in this tissue, sufficient TAg is required so that constitutive levels occurring in vitro can interfere with the normal processes of differentiation and growth control.
Is insufficient to express the level of Such findings are consistent with the hypothesis that the occurrence of thymic hyperplasia in vivo is enhanced by the presence of hepatitis infection in mouse colonies. Such infections cause increased production of interferons in the thymus, pushing T-antigen levels above the ineffective threshold. These results indicate that it is necessary to avoid the inappropriate expression of TAgts levels in all cells in the body (not just those exposed to inducers), in this case as a result of mouse colony disease. Further support the idea.

The central nervous system-derived cell line as another illustration of the present invention is a potential use of the cell line obtained by gene transfer as an imaginary precursor of glial tumors and as a means of studying differentiation control It is particularly interesting as an indicator of sex. This cell line
It can express astrocyte-specific antigens when grown under certain tissue culture conditions, but can be induced to express a fibroblastic phenotype under other tissue culture conditions. The reason that this central nervous system cell line is characterized as an imaginary glioma precursor is that human gliomas
A cell that is clearly derived from an astrocyte;
Antigens can be classified into two categories: cells that do not express GFAP and instead express fibronectin (FN).
It is in the idea. It has been demonstrated that cloning of the GFAP expression system results in the generation of a GFAP negative cell line that expresses fibronectin, and therefore, cells that express fibronectin, which are not correlated with any known central nervous system (CNS) glial cells, It is suggested that the origin can be traced to the pedigree. Several experiments indicate that a subset of GFAP-positive astrocytes, considered rare, may also express FN (most astrocytes do not), potentially implicating these findings. ing. The cell lines described in this document can be grown by growth in fetal bovine serum.
It is possible to switch from GFAP positive phenotype to FN positive and GFAP negative phenotype. The ability to manipulate the differentiation of this cell allows for an assay system suitable for the purification of specific molecular signals that induce differentiation along the FN-positive, GFAP-negative pathway.

Another cell line generated by the method of the present invention is derived from the pancreas. The small number of cells in this system express insulin naturally under all conditions of growth, and some cells in the system can also be labeled with a monoclonal antibody (A2B5) that is thought to label pancreatic islet cells. It is not yet known whether variable expression of these markers in cell lines failed to create a differentiation-inducing microenvironment appropriate for tissue culture conditions.

Other matters (and related aspects of the invention) specifically exemplified in the work described herein are specifically mentioned in the following examples. The following non-limiting examples illustrate the principles of the present invention. As used below, "mouse X" refers to the Xth mouse from the experiment described first.

Example 1 Construction of pH-2KbtsA58 Gene Construct Recombinant pH-2KbtsA58 (see FIG. 1) adds the 5′-promoter element of the H-2Kb gene to the early region coding sequence from SV40 mutant tsA58. Made by Plasmid pH-2Kb as an EcoRl-Nrul fragment with a promoter fragment of about 4.2 kb (this is Dr. Andrew Mellor, MRC London)
Provided by). This plasmid contains an EcoRl fragment containing the genomic sequence encoding the H-2Kb gene from cosmid 88H8 (Weiss et al., Nature 1).
983, 301, 671-674 (Weiss et al, Nature 1983, 301, 67
1-674)) was cloned into plasmid pBR327. tsA58 DNA was obtained from Tegtmeier, 1975 J Virology 16, 168-178 (Tegtmeyer, 1975 J. Vir.
ology 16,168-178). In this example, SV4
The 0tsA58 coding sequence was developed by Dr. Hartmut Land (of the Imperial Cance, London).
r Research Fund, London)) provided by pUCSV4
It was isolated as a Bgll-BamHl fragment of about 2.6 kb from 0tsA58. This plasmid was derived from Kpnl (nucleotide 294) derived from tsA58 DNA encoding the T antigen coding sequence to BamHl
The fragment of (nucleotide 2533) was converted to Kpnl and Bam of pUC19.
Created by insertion at the Hl site. The Bgll site is
Klenow fragment of DNA polymerase I
blunt-ended using t). Both fragments are EcoRl and Ba
It was bound to an equimolar number of puc19 digested with ^mHl . The ligated product was transformed into JS4, a recA ^- derivative of E. coli MClO61 (Casadavan and Cohen 1980 J. Mole Viol 138, 179-207; Cedibi et al. 1987 cell 50,
379-389 (Casadaban and Cohen 1980 J. Mol. Biol. 138,
179-270; Sedivy et al 1987 Cell 50,379-389)),
Ampicillin colonies were isolated. To determine if the promoter fragment successfully fused to the T antigen coding sequence, DNA minipreps were made from isolated colonies and digested with various restriction endonucleases and analyzed.

Generation of Transgenic Mice Containing H2K ^b -Tagts Fusion The above H-2K ^b -TagtsA58 plasmid was digested with EcoRl and Sall to prepare a DNA fragment without vector sequence. These DNA fragments were separated on an agarose gel and TE buffer (10 mM
M Tris, pH 7.5, 0.2 mM EDTA)
At a concentration of ml, fertilized single cell mouse eggs were injected into the male pronucleus. Oocytes that survived microinjection were replaced by Wagner et al. (198
1) PNA, 78, 5016 (Wagner et al (1
981) Transferred to pseudopregnant females as described in PNAS78,5016). The ova are from the CBA x C57BL / 10 cross. Mice were obtained from MRC breeding colonies and housed in environmental controls maintained on a cycle of 10 hours dark and 14 hours light. Oocytes in the rearing female were allowed to develop to the limit.

Analysis of Transgenic Mice At 7-14 days of age, each child was analyzed to see if it had the transgene. First, DNA prepared from a small portion of the tail was analyzed on a slot blot. Sambrook et al. “Molecular Cloning” (Cold Spring Harbor 1989 (Sambrook et al “Molecular Cloning” (Cold
Spring Harbor 1989)).
Genomic DNA was separated from the 0.1-0.15 cm section. The resulting nucleic acid pellet was washed once with 80% ethanol, dried, and resuspended in 200 μl 10.0 mM Tris, pH 7.4, 1 mM EDTA. The filter with specific ³² p-labeled fragment from the SV40 large T antigen to confirm the presence of the construct by hybridization. Feinberg & Vogelstein
Probes were prepared by random priming method. tail
Southern blot analysis by digesting 10 μg of DNA with BamHl confirmed the integrity of the TAg gene. Digested DNA
Fractionation on a 0.8% agarose gel, transfer to ZetaBind ^™ (Zeta Bind ^™ (Biorad)), and published methods (Sambrook et al.
(as described in mbrook et al)) and hybridized with an SV40 specific probe. ³² p for TAg gene
The blot was probed with a labeled probe. All manipulations were performed at the conditions recommended by the manufacturer or by standard protocols as described in Sambrook et al., Supra. Slot blot showed that 34 of 88 mice had the chimeric gene. The copy number of this fusion gene present in mouse DNA is 1
There were 1-15 copies per cell.

Animals containing the H-2K ^b -TAgtsA58 fusion gene developed normally except for the development of thymic hyperplasia. Expressed TA
There appeared to be a clear correlation between g mRNA levels and the rate of onset of thymic hyperplasia. Both thymus lobes spread evenly, and as many as 10 ⁷ thymus-derived cells were injected (n.c. or intraperitoneally) into native recipient mice, but not into host mice. The fact that it did not cause a tumor indicates that the observed thymic enlargement was not due to neoplastic transformation. In addition, as described below, almost all stromal cell lines from these expanded thymus appeared to be conditional on growth in culture. Thymic hyperplasia may be due in part to the presence of a mouse hepatitis virus infection in animal colonies that would cause interferon production in infected animals. Thus, already high levels of endogenous class I antigen expression in thymic tissue may have exacerbated hepatitis infection, leading to higher levels of TAgtsA58 expression in vivo than in other tissues of the body. In one mouse strain that expressed only a single copy of the hybrid gene, the development of hyperplasia took longer (6 months for heterozygotes and 3-4 months for homozygotes).

A large amount of TA, such as cells grown in tissue culture described below,
When gtsA58 is transcribed (in connection with the presence of multiple gene copies in mouse DNA), it appears that even at permissive temperatures, TAgtsA58 has a marginal effect on growth promotion. In one of the mice, where only one copy of TAgtsA58 is present in the genome, only thymic hyperplasia occurred only a few months later, and the mouse was able to reproduce normally and effectively, and H2K ^b -Transmitted the TAgtsA58 fusion to progeny.

Analysis of Fibroblasts from H2K ^b -TAgtsA58 Transgenic Mice The following is important for selecting mice that show low levels of transgenic expression in vivo for the mice to function in accordance with the principles of the present invention. Shows sex. As previously mentioned, this selection method is the exact opposite of the standard method for transgenic animal selection, which requires high levels of in vivo transgenic expression to maximize the potential for inhibiting normal in vivo development. The method of the present invention is the opposite of such a standard method.

To demonstrate that expression of H2K ^b -TAgtsA58 in the tissues of transgenic mice allows the generation of a conditionally immortalized cell line, the growth of skin fibroblasts was examined. First, the mice were sacrificed by cervical dissection, the skin and hair were sterilized with ethanol, the hair was shaved off, and the skin was separated by several centimeters square to obtain fibroblasts from five different mice. The skin was minced with a sterile dissector and digested at 500 units per ml of collagenase in Leibovitz's L-15 medium for 2 hours at 37 ° C. And 3000 per ml
Trypsin is added to the final concentration of the unit and the tissue is further incubated at 37 ° C.
Maintained for 15 minutes. After this incubation, a soybean trypsin inhibitor, also prepared in L-15 (10 / ml
(00 units) and DNAse (15 units per ml) were added to terminate the enzyme digestion. The tissue was made up to a volume of 5 ml and gently moved up and down with a sterile plastic pipette for a total of 20 times to grind. Non-separated tissue clumps are sedimented, and the cells contained in the supernatant are first centrifuged and washed, and Dulbecco's Modified Eagles containing 2 mM glutamine, 10% fetal calf serum, and 100 U / ml recombinant mouse gamma interferon.・ Medium (Dulbecco's Modified)
Eagle's Medium). H2K ^b -TAgtsA58 in all cases where cells were obtained from mice with and thus created cultures effectively grown in tissue culture flasks. Cells from normal controls of the same age were also prepared and grown in the same manner. Cells from these normal mice aged after a short time, as described below.

Cultures generated from H2K ^b -TAgtsA58 fusion transgenic mice were cultured for 8-12 weeks before testing their growth conditions.
Grow at 33 ° C. in the presence of 100 U / ml gamma interferon. Long before this time, all cells from mice that did not contain the H2K ^b -TAgtsA58 fusion construct,
The division stopped after a crisis, as expected for non-immortalized fibroblasts. All fibroblast cell lines derived from H2K ^b -TAgtsA58 fusion transgenic mice, cell growth was inhibited when placed in non-permissive conditions. FIG. 2 shows that 1000 cells were plated on a 6 cm dish in DMEM + FCS without interferon.
Plates for 1 hour, then transfer to medium with or without 100 g / ml mouse gamma interferon and grow at 33 ° C or 39.5 ° C for 14 days, during which time the medium is grown for 2 weeks.
The results of a colony formation assay performed at different times are shown.
Pre-plating for 24 hours in normal medium is the same as initial plating for all cultures. After 14 days, the culture was stained with 2% methylene blue, 50% ethanol: water and the number of colonies obtained was counted. As shown in FIG. 2, cell growth under fully permissive conditions (ie, 33 U, 100 U / ml mouse gamma interferon) was greater than under any non-permissive conditions.

A detailed analysis of dermal fibroblast cultures for growth conditionals revealed three groups of cultures by the ability of cells to grow in fully permissive, semi-permissive and non-permissive conditions, but for this purpose The permissive condition is defined as growth at 33 ° C in the presence of IFN gamma, and the semi-permissive condition is defined as 33 ° C without IFN gamma or growth at 39.5 ° C in the presence of IFN gamma, non-permissive condition Is defined as growth at 39.5 ° C without IFN gamma.

In the first group of cultures, growth was completely conditional and only occurred under permissive conditions. Grown at 39.5 ° C and / or
When grown without IFN gamma, cell division does not occur in either the standard growth assay or the colony formation assay. Thus, these fibroblasts were derived from previous studies in which rat embryonic fibroblasts were conditionally immortalized with tsA58TAg by retroviral infection ((Jat and Sharp, 198
9, Mor cell viol 9, 3093-3096 (Jat and Sha
rp 1989, Mol. Cell, Biol., 9, 3093-3096)). In these earlier studies, tsA58TAg
Fibroblasts conditionally immortalized using retrovirus-mediated gene insertion to produce cell lines that express
It has been shown to continue breeding only if maintained at acceptable conditions. When the temperature is changed to non-permissive conditions, the fibroblasts rapidly develop the senescent phenotype expressed by normal fibroblasts that have been grown in vitro for long periods of time. Mouse H2
All cultures from different individuals within the ^Kb- TAgtsA58 strain produced identical results.

In the second group of cultures, the best growth was obtained under fully permissive conditions, with a lower degree of growth under semi-permissive conditions,
No growth under non-permissive conditions. The third group had the best growth under fully tolerated conditions and had the slowest growth under completely non-permissive conditions, but the cells completely stopped growing even under non-permissive conditions. Did not.

The growth condition observed in fibroblasts from transgenic animals correlated with the level of tsA58TAg expressed by these cells. In all cultures, tsA58TAg levels were reduced by shifting up the temperature and / or removing IFN. Interestingly, the most conditioned culture (derived from the mouse H2ts6 progeny) was IFN at 33 ° C.
Low levels of TAg were detected even when these cells were grown in conditions where they did not grow without gamma. This finding is described in detail in the following example.

To determine whether a conditionally immortal cell line is completely immortalized by the introduction of a constitutively active differentiation-inhibiting gene, a portion of fibroblasts isolated from H2ts6 mice was
Infected with a retrovirus expressing the wild-type SV40T antigen and the neomycin resistance gene (Jat & Sharp, Jay Byrol 1986, 59: 746-750 (Jat & sh)
arp, J. Virol., 1986, 59: 746-750)). Successfully infected cells were selected by growth in G418 antibiotic. When these cells were switched to non-permissive conditions, they continued to grow. Thus, these experiments indicate that the cell line can be switched from a conditional to a non-conditional growth state when it is considered desirable.

Analysis of H2K ^b -TAgtsA58-Derived Cell Lines Analysis of Transgenic Mouse Thymocyte Cell Lines (1) This study demonstrates that hyperplasia in vivo, even in a single instance where the gene construct used inhibits normal development This shows that the expanded cell type remains conditional in its in vitro growth. (2) This study further suggests that thymic epithelial cells from H2ts6 mice express a family of intermediate filament proteins normally expressed in vivo by these cells, thus making these cells their normal counterparts. Figure 4 shows that it is a potentially suitable source for purification of the expressed protein. (3) This study also shows that the thymic epithelial cell line from H2ts6 mice is capable of rosetting T lymphocytes, and thus is a suitable cell for biochemical dissection that could be considered a study of normal cell interactions. Can be typed.

For the development of thymic hyperplasia in many H2K ^b -TAgtsA58 fusion transgenic mice, it was important to characterize the growth of thymus-derived cells. For this purpose, a total of 15 minutes of collagenase plus an additional in the presence of additional trypsin
Thymocytes were prepared for culture as for fibroblasts, except that the time during which the enzyme was added was limited to 15 minutes. And these cells, like skin fibroblasts,
Grow until confluent flasks were obtained, after which clonal cell lines were isolated by limiting dilution single cell cloning.

The cell line isolated from the thymus of the H2K ^b -TAgtsA58 fusion transgenic mouse was conditional on growth and was inhibited from growing when grown at 39.5 ° C. without interferon. The in vitro conditioning of these cell lines results in the expression of sufficient levels of T antigen such that endogenous levels of class I antigen expression in this tissue can also interfere with the normal processes of differentiation and growth control. Is insufficient. Such findings are consistent with the hypothesis that the cause of thymic hyperplasia in vivo is probably due to the presence of hepatitis infection in mouse colonies. Such infections cause increased production of interferons in the thymus, pushing T-antigen levels above the ineffective threshold. These results indicate that the strong constitutive promoter causes inappropriate expression of TAgts levels in every cell in the body, not just in cells exposed to the inducer, as a result of mouse colony disease. It further supports the importance of the use of a sex, activatable promoter.

Two of the thymus-derived cell lines were characterized antigenically, and they were found to express cytokeratin by staining with a LE61 pan-antikeratin monoclonal antibody. Since keratin is specifically expressed in epithelial cell populations, labeling these cells with the LE61 antibody indicates that these cells are thymic epithelial cells.

The cytokeratin-positive thymic epithelial cell line described above specifically bound T lymphocytes as detected by a standard rosetting assay. Stromal cells were mixed with freshly isolated unsorted thymocytes from non-transgenic BALB / c mice at a ratio of 1:12. Cells were kept in small volumes (200 microliters) and incubated on ice for 1 hour. The mixture was then centrifuged at 200 g for 5 minutes, the pellet was gently resuspended in 1 ml of PBS, and the rosette was counted using a hemocytometer, with three or more thymocytes attached to stromal cells as one rosette. . In this assay, the cytokeratin + thymic epithelial cell line efficiently formed rosettes with thymocytes.

Analysis of Putative Glioma Progenitor Cells To obtain cell lines of the central nervous system, Noble et al.
4, Jay Neurosai 4, 1892-1903) (Noble et
al (1984, J. Neurosci. 4, 1892-1903), cells were isolated from the cerebral cortex of mouse 11. Mice were 3 weeks old at the time of dissection. Cells are grown in chemically defined media (Bottenstein and Sato,
1979, Plock Natl Acad Sai USA
76, 514-517 (Bottenstein and Sato, 1979, Proc. Natl.
Acad. Sci. USA 76, 514-517)) with 10 ng / ml platelet derived growth factor [BB homodimer (supplied by British Biotechnology) and 10 ng / ml PDGF AA homodimer. (Supplied by Chiron Corporation)] and 10 ng / ml basic fibroblast growth factor (Boehringer-Mannheim)
(Supplied by Co., Ltd.). Cells were grown at initial passage (passage) and then cloned by limiting dilution. Ten clones were isolated and characterized for antigen expression. Among them, one clone is a collagen fiber acidic protein (GFAP) which is a cytoskeletal protein specifically expressed by astrocytes in the central nervous system (Bigami et al., 1972 Brainless 43, 429-435 (Bignami , et al,
1972 Brain Res 43, 429-435)). Dakopats Limited
patts Ltd) and an appropriate fluorescent second layer antibody (Southern Biotechnology)
technology) (purchased from Technology).

To examine the differentiation potential of the cells for GFAP-expressing clones, the cells were re-cultured on poly-L-lysine-coated glass coverslips and treated with various substances to induce differentiation. Of particular interest is the effect of fetal bovine serum, which induces cells
A GFAP-phenotype was generated. GFAP-cells did indeed express the extracellular matrix protein fibronectin (FN), which has been reported to be expressed only in some low-defined astrocyte subpopulations of central nervous system glial cells. Several experiments suggest that the parent cell line expressed both GFAP and FN, but that in the absence of serum, the parent cell could exhibit the GFAP + FN- phenotype.

Separating regulatable cells between the GFAP + and GFAP-FN + phenotypes is of great interest in light of recent studies of antigen expression in human gliomas. The morphological classification of human gliomas generally states that these cells have a close lineage relationship with normal glial cells of the central nervous system,
Extensive antigen analysis of cells derived from gliomas is inconsistent with the morphological classification of these tumors (Kennedy et al., 1987 Neuropath apple neuroviol 13, 327-347 (Ke
nnedy et al 1987 Neuropath Appl. Neurobiol. 13,327−
347)). Very importantly, gliomas fall into two antigen categories, the first being the GFAP + phenotype and the second being GF
Previous studies have shown that it can be classified as one of the AP-FN + phenotype. Kennedy et al. Reported that GFAP-FN
+ Most glioma-derived cell cultures with glioma phenotype
It was found to occur in 90%. Although it has been shown that clones from GFAP + gliomas may be GFAP- and FN-, the origin of these cells is not yet known (Westfar et al., 1988, Cancer Research 48, 731-740).
(Westphal et al, 1988, Cancer Research 48, 731-74
0)).

The results for human gliomas show that astrocytes (GFAP +)
There is a significant possibility that progenitor cells with the potential to differentiate along both the and glial cell (GFAP-FN +) pathways are present in the nervous system. The cells we have isolated are the first such cells to be clearly isolated from the brain in a way that allows a close examination of this possibility. Numerous studies of antigen expression in glioma cells and the agreement of the antigenic phenotype that cells isolated from mouse 11 can express clearly suggest that this cell line is a candidate for glioma progenitor cells. ing.

Analysis of Pancreatic Cells Pancreatic cells isolated from mouse 11 pancreas in the same manner as cortical cells and grown and cloned in the same manner as cortical cells were pre-characterized in tissue culture. A very small portion of the cells of the clones examined very closely express insulin (as recognized by anti-insulin antibodies purchased from ICN) and can be labeled with monoclonal antibody A2B5 (National Institute of the United States). Dr. Marshall Neilenberg of Health
shall Nirenberg of the National Institute of Healt
h, USA), both of which are markers for pancreatic islet cells (Eisenbarth et al., Proc. Natl.
9, 5066-5070 (Eisenbarth et al, Proc, Natl, Acad, Sc
iUSA, 79, 5066-5070)).

It is unfortunate in retrospect that both cortical cells and pancreatic clones were isolated from mouse 11, and analysis performed much later revealed that they were one of the least conditional of transgenic mice . However, preliminary results indicate that cortical and pancreatic cells may be more conditional than fibroblasts. More importantly, the mouse
Neither the brain nor the pancreas showed major developmental abnormalities, indicating that the levels of TAgtsA58 that may have been expressed in these cells were insufficient to interfere with in vivo differentiation. Alternatively, placing these cells in tissue culture conditions may have expressed higher levels of class I antigen than in vivo.

One of the transgenic mice (mouse No. 6) made with the H2K ^b tsA58 construct grew well and produced litters, all of which had the original phenotype. . In all of the following examples, the transgenic animals used were
It was a heterozygous offspring of what was called the H2ts6 strain (mouse No. 6).

Example 2 Cells were prepared from one heart of the offspring of mouse 6 in the same manner as fibroblasts were generated from the skin of another mouse. Mouse used as material for preparing heart tissue ("Daughter of 6"
Did not show any obvious abnormalities in organ size when dissected.

Cells from six daughters (identified as suitable heart-derived fibroblasts) were grown at 33 ° C. for 4 weeks in the presence of recombinant mouse gamma interferon. Cells were cultured overnight on poly-L-lysine-coated glass coverslips at 33 ° C. in DMEM + 10% fetal calf serum for experimental analysis. The next day, cells were switched to growth at 33 ° C or 39.5 ° C in the presence or absence of gamma interferon. After growth for 3 days in permissive or non-permissive conditions, bromodeoxyuridine was added for 24 hours, the cells were fixed (with the protocol supplied by Becton Dickinson) and anti-bromodeoxy (purchased from Becton Dickinson). The cells were stained with a uridine antibody and then labeled with a rhodamine-conjugated secondary antibody (purchased from Southern Biotechnology) for the last 24 hours that had been involved in DNA synthesis. As shown in FIG. 3, cells grown at 33 ° C. in the presence of interferon were compared to cells grown at 39.5 ° C. without interferon during the labeling period.
Had 20 times the DNA synthesis. Cells grown in semi-permissive conditions exhibited intermediate levels of DNA synthesis well suited for survival.

Example 3 (1) This study demonstrates the principle that cells from H2ts6 mice are conditionally immortal and follow the normal pathway of terminal differentiation when switched from permissive to non-permissive conditions. (2) This study further demonstrates that sub-functional levels of oncogene products can be expressed, thus confirming the principle that oncogene product levels can be expressed without interfering with normal developmental pathways. I do. (3) Furthermore, this study demonstrates the need for fine-tuning the differentiation inhibitory activity of oncogene products in order to maintain activity below levels that interfere with normal development.

A culture of dermal fibroblasts from H2ts6 mice was generated as described in Example 1, which exhibited a conditionally immortalized phenotype as detailed in the previous example.

[0103] Western blot analysis of T antigen expression in cultures of fibroblasts from H2ts6 mice showed that expression was clearly higher in the presence of IFN gamma, but at 33 ° C in the presence or absence of IFN gamma. It showed relatively low levels of T antigen expression.
This observation indicated that dramatic changes in cell growth could be observed as a result of small changes in the level of this gene product. To test this possibility, cell growth and colony formation
A dose response analysis titrated against IFN gamma concentration was performed.

[0104] Fibroblasts from H2ts6 mouse progeny showed enhanced cell growth with levels of IFN gamma as low as 1 U / ml. Analysis by colony formation and cell number analysis showed that adding 100 U / ml IFN gamma to these cultures only increased the frequency of colony formation by a factor of 3.5 compared to that seen in the presence of 1 U / ml. , Showed only a 40% increase when 10 U / ml was added. The difference in TAg levels at different doses of IFN gamma was not significant, with 1 U / ml giving a 2.5-fold increase over basal levels and 100 U / ml giving about a 6-fold increase over basal levels.

Example 4 Generation of Astrocyte Lines This example illustrates four points of use of the animals of the present invention. (1) Astrocyte lines representing defined differentiated cell types were generated from the central nervous system, a tissue with essentially no endogenous levels of class I antigen expression and no in vivo production of mRNA. Thus, we show that in vivo transcription of oncogene constructs is not a prerequisite for cell line generation. (2) Astrocyte lines express cell type-specific markers associated with normal cell types, thus demonstrating the potential utility of cells from H2ts6 mice as a source of cell type-specific protein purification . (3) The astrocytic cell line produces mitogenic (mitogenic) activity known to be expressed by the cell's normal counterpart, so that the astrocytic cell line from the H2ts6 mouse can Has the ability to promote the division of mitogens, and thus represents a potential source for the purification of mitogenic (mitogenic) factors. (4) Finally, the astrocytic cell line is to first grow brain cells under normal (non-permissive) tissue culture conditions, then purify the target cell type, and then grow the target cells under permissive conditions. Produced by Thus, this indicates that it is not even necessary to conditionally immortalize cells at the initial dissection since the oncogene product function is initiated after the cells have been grown in tissue culture for a predetermined period of time.

Standard Procedure (Noble et al., 1984, Jay Neurosai, 4: 1892-1903: Noble and Murray, 1984;
Embo Jay, 3: 2243-2247 (Noble et al., 1984, J.
Neurosci., 4: 1892-1903; Noble & Murray, 1984, EMBO
J., 3: 2243-247)), a culture of cortical astrocytes was prepared. Briefly, cortex from newborn H2ts6 mice was treated with 0.25% of the same amount of 0.25% collagenase in L-15 medium.
Separated into single cells by enzymatic digestion of the tissue with trypsin. At 37 ° C. in DMEM containing 10% fetal calf serum, glutamine 2 mM and gentamicin 25 microgram / ml,
The culture was grown. After 7-10 days, the culture was placed on a rotating platform at 37 ° C. overnight and spun at a speed slightly slower than the speed at which the medium became foamy (ie, about 60-75 rpm). As previously described (Nobel et al., 1984), this procedure yielded 95% of these cells with astrocyte-specific cytoskeletal proteins.
It produces a culture that is 95% pure astrocytes in view of the expression of GFAP (collagenous fibrillary acidic protein).

After purifying astrocyte cultures to> 95% purity, a clonal cell line was generated by first converting the cells to 33 ° C. in the presence of gamma interferon. Then, a retrovirus harboring the bacterial β-galactosidase gene and the neomycin resistance gene, and a standard infection protocol using this virus (Price et al., Proc. Natl.
tal, Proc. Natl. Acad. Sci. USA, 1987, 156-160)). One day after infection, 0.
Remove cells from flask by incubation with 25% trypsin (see Noble et al., 1984, supra),
Re-plated in medium containing G418 antibiotics. From the selection conditions, resistant colonies of cells were generated, and 10 clones were randomly selected for continuous research. In this manner, clonal cell lines were readily generated, with seven out of ten cell lines constitutively expressing collagen fibrillary acidic protein (GFAP), an astrocyte-specific marker in the central nervous system.

To determine the ability of astrocyte lines to produce mitogenic activity normally associated with these cells,
Progenitors of oligodendrocyte type 2 astrocytes (O-2A) were isolated from the optic nerve of 7-day-old rats and plated on astrocyte monolayers. In an earlier experiment (Noble and Murray, 1984), astrocytes, but not meningeal cells or fibroblasts, could stimulate O-2A progenitor cell division in vitro and are regulated by astrocytes O-2A progenitor cells stimulated to divide by the prepared medium are converted to astrocyte monolayers, astrocyte conditioned medium or platelet-derived growth factor (mitogens produced by these astrocyte monolayers (mitogenesis). factor))
Has been shown to express a particular bipolar morphology that is only seen when these progenitor cells are grown.
O-2A progenitor cells grown on a monolayer of a clonal astrocyte line derived from the transgenic mouse strain H2ts6 grew on non-transgenic astrocytes with respect to division and expression of the expected bipolar morphology I could not distinguish it from the thing.

Example 5 Central Nervous System Glial Progenitors This example illustrates the following: (1) Immediately immortalize cells derived from the central nervous system having characteristics of novel progenitor cells by cell growth under permissive conditions, and the cells thus grown are usually associated with nuclear oncogene expression. There is a possibility of preventing differentiation. Thus, this example further demonstrates the ability to produce immortalized cell cultures from body tissues without detectable in vivo expression of the transgene. (2) Cell division requires the presence of appropriate growth factors, which indicates the potential utility of cells from H2ts6 mice as an assay system useful for growth factor purification. (3) Progenitor cells from the H2ts6 mouse can be induced to differentiate by switching the cells from permissive to non-permissive conditions, thus indicating that these cells have a potential to grow new progenitor cells. Indicates potential utility. (4) The progenitor cells grown according to the method of the present invention, when grown under permissive conditions, retain the ability to undergo normal differentiation if the cells are exposed to a medium adjusted with defined molecular factors or cell sources. I do. Thus, this example further illustrates the potential utility of cells from H2ts6 mice for use in assay systems that allow for the purification of factors that induce cell differentiation.

Cells were derived from mice on day 18 of development, platelet-derived growth factor AA homodimer (Kilon Corporation) at 10 ng / ml, basic fibroblast growth factor (Kilon.
Corporation) at 5 ng / ml and IFN gamma at 20 U / m
l containing chemically defined medium (Ruff et al., 1983, Nature, 303: 390-396 (Raff et al., 1983, Nature, 303: 39).
Cortical cells were isolated as described in Example 3, except that they grew on components (as described in 0-396). Cells could be rapidly passaged and passaged cells maintained at the described conditions showed no evidence of differentiation to defined glial cell types. The culture was performed using an A2B5 monoclonal antibody (Eisenbarth et al., 1979, Proc Natl Acad Sai, USA), 76: 4913-4917 (Eisenbarth et al, 1979, Proc.
c. Natl. Acad. Sci. USA, 76 :: 4913-4917), including bipolar cells that can be labeled with O-2A in optic nerve cell culture.
Similar to progenitor cells (e.g., Ruff et al., Nature, 1983, 303: 390-396; Noble and Murray, 1
984, Embo Jay, 3: 2242-2247 (Raff et al., Nat.
ure, 1983,303: 390-396; Noble & Murray, 1984, EMBO
J., 3: 2243-247)). Cultures were also performed by antibodies against vimentin intermediate filaments (antibodies from Dako-Patts, Ltd.) and by SSEA-1 (Gouy et al., Nature, 1981, 292: 156-1).
58 (Gooi et al., Nature, 1981, 292: 156-158) contained another group of novel cells labeled with antibodies to the stage-specific embryonic antigen-1). These new cells exhibited very primitive morphology and were small, round cells with few protrusions and cytoplasmic extensions. Previous experiments on cultures made from embryonic rat cortex showed similar cells, both of which were consistently passaged (regardless of the medium in which the passaged cells were replated). (Passage) to differentiate into astrocytes or oligodendrocytes. In contrast, cells derived from H2ts6 mice maintained under the above growth conditions were easy to passage repeatedly without differentiation.

[0111] Cortical cells derived from the cortex of H2ts6 mice could be induced to differentiate by several in vitro manipulations. In all cases, the culturing Tomoeda glial cells (which, Tomoeda glial cells A2B5 + SSEA-1 ^- because it is, A2B5 + was thought to be derived from cells) and astrocytes (which, stellate Cells often
SSEA-1 ⁺ but always A2B5 ^- a since yielded was considered) to be derived from the SSEA-1 ⁺ cells. In both cases, cell morphology changed dramatically. Oligodendrocytes express their normal multipolar form, and monoclonal antibodies to galactocerebroside (Rancit et al., 1982, Proc Natl Acad Say, USA, 79: 2709-2713 (Ranschte)
t al, 1982, Proc. Natl. Acad. Sci. USA, 79: 2709-271.
3)) could be labeled. Astrocytes form in the case of differentiation of the cells changed dramatically, SSEA-1 ⁺ cells appear small primitive is the cell body large, SSEA-1 with a spread wide film ⁺
Replaced by cells. Cells differentiated into astrocytes not only resembled astrocytes, but also expressed GFAP.

First, the elimination of PDGF and FGF leads to differentiation and cell death, which therefore requires that the oncogene-expressing cells require the continued presence of appropriate growth factors in order to continue to grow. Is shown. Second, cells maintained in the presence of growth medium and growth factors also differentiated when IFN gamma was removed from the medium (thus terminating TAgts expression). These results indicate that TAgts expression is required to prevent differentiation even when cells are grown under conditions that allow oncogene-expressing cells to continue to grow in an undifferentiated state. . Third, cells grown in the presence of PDGF and bFGF under fully permissive conditions (ie, at 33 ° C., + IFN gamma) were purified cortical astrocytes (Noble et al., 1984, J. Neurosy, 4: 1892-1903 (Noble et al., 1984, J. Neurosci.,
4: 1892-1903) were induced to differentiate upon exposure to the medium conditioned by
The potential utility of H2ts6 progenitors for use in assay systems for the detection and purification of differentiation inducers was demonstrated. Fourth, cells grown in fully permissive conditions in the presence of PDGF and bFGF were exposed to 10 ng / ml transforming growth factor-β (British Biotechnology) or 2 ng / ml hairy Differentiation is induced by exposure to somatic neurotrophic factor (Synergen), and thus demonstrates the responsiveness of progenitor cells to defined differentiation-inducing agents known to be present in the central nervous system and further induces differentiation The potential utility of H2ts6 progenitor cells for use in assay systems for agent detection and purification has been further demonstrated.

Example 6 Endothelial Cells (1) This example shows that H2ts6 mice can be used to generate cell lines of endothelial origin. (2) This example further demonstrates that endothelial cell cultures generated from H2ts6 mice secrete a novel differentiation promoting activity known to be secreted by normal endothelial cells. Thus, this example further demonstrates the potential utility of H2ts6-derived cell lines as a source material to allow for the post-purification of molecules that express unique biological activities.

[0114] Endothelial cell colonies were prepared as follows. Two mature mice (2-3 months of age) were put into a CO ₂ coma and their heads were cut off. Gentamicin at 25 micrograms / m
The brains were washed with Leibowitz L-15 medium containing l and placed in fresh L-15. Each brain was placed in a 30 mm Petri dish containing several mls of L-15. The cerebellum and other white matter cords (callosal and eyeballs) were cut off. The meninges were removed cleanly. Finely chop the remaining gray matter with a sterile dissecting sword,
Once passed through a 19 needle gauge and incubated with 0.1% collagenase: dipase (BCL) in L-15 at 30 ° C. for 60 minutes. Spin the tissue at 1000g for 10 minutes at 4 ° C,
The supernatant was discarded. 20 ml of 25% BSA in L-15 was added, mixed well without bubbling, and spun at 2000 g for 20 minutes. The buoyant layer of the tissue was carefully removed with the supernatant without disturbing the small pellet. Mix supernatant and tissue again for 20 minutes
Rotated at 00g. This time, discard the tissue layer and the supernatant.
Individual pellets were suspended in 10 ml of 0.5% BSA in L-15,
The pellet was spun at 1000g for 10 minutes at 4 ° C to wash. The pellet was suspended in 0.1% collagenase (in L-15): dispase and incubated at 30 ° C for 2 hours. After incubation, DNAse was added to a final concentration of 10 micrograms / ml, and the resulting capillary-containing tissue was spun at 1000 g for 10 minutes at 4 ° C. Pellets C
The suspension was gently resuspended in 1 ml of DMEM without a-Mg, layered on a 10 ml Percoll gradient and spun at 1000 g for 10 minutes at 4 ° C. (5 parts of isosmotic Percoll [9 parts of Percoll and PBS1 without 10XCa-Mg
5 parts of 1 × PBS, and rotated at 26,000 g for 1 hour to prepare a linear gradient of 50% Percoll (Pharmacia) in PBS without Ca-Mg. ) The top half of the tube contained cell debris and single cells. In the lower half, there were red blood cells that looked like a red ring, and a capillary just above the ring. This layer was carefully removed, suspended in 15 ml of L-15 and spun at 1000 g for 20 minutes at 4 ° C. The supernatant is discarded and the capillaries are grown in growth medium (2 mM glutamine, Bogel et al. (Proc. Natl. Acad. Said, 1978, 75: 2810-2814 (Vogel et al. (Proc. Natl. A.
cad. Sci., 1978 75: 2810-2814) 20% plasma from serum as described in 10), 10 IU / ml heparin (Sigma), 5 ng.
/ ml basic FGF (Kilon Corporation) and 20
DME containing 4.5 g / L glucose supplemented with U / ml IFN gamma
Suspended gently in M. Capillaries were cultured in Bitorojen (Flow Lab) (Vitrogen (Flow Lab)) 50% occupancy in the coated 96-well plates were incubated with 7.5% CO _2. The medium was changed three days later and then every two days. On day 3, single-capillary wells were labeled and grown to confluence. Endothelial cells generated from these capillaries grew as colonies with tight borders. These cells were gently trypsinized (0.025% in DMEM without Ca-Mg containing 2 mM EDTA).
Trypsin, 3 minutes at 30 ° C) and 2% (w / v) gelatin (Difco) made in sterile tissue culture grade water.
falcon pre-coated with gelatin by incubating the growth surface of the flask with
Subculture was achieved by replating in a 75 cm ² flask. Immediately before use, the gelatin was aspirated and the flask was washed with medium. Unlike normal endothelial capillary cells from mouse brain, these cells can be passaged repeatedly.

[0115] Endothelial cell cultures were examined to determine if these cells produced a novel differentiation regulating activity that was produced by their normal counterparts. The differentiation of O-2A progenitor cells into type 2 astrocytes is
It requires the presence of at least two suitable inducers, these are ciliary neurotrophic factors and unknown factors found in the matrix of endothelial or conjunctival cultures (Lilien.
Andruff, 1990, Neuron, 5: 111-119 (Lillie
n & Raff, 1990, Neuron, 5: 111-119)). Our own studies indicate that factors that induce astrocyte differentiation in cooperation with ciliary neurotrophic factors are secreted from various normal endothelial cells but not from other cell types. ing. Endothelial cell lines generated from H2ts6 transgenic mice, like all non-transgenic endothelial cells tested so far, have the potential as this source of differentiation stabilizing activity.

Assays used to recognize endothelial cell-derived factors that cooperate with ciliary neurotrophic factors are based on standard methods (eg, Raff et al., 1983, Nature, 303: 390-396).
(Raff et al., 1983, Nature, 303: 390-396))
A culture of optic nerve cells was made from rat nerves of the day, and these cells were cultured for 24 hours in a confluent culture of bovine aortic endothelial cells (Ruff et al., 1983, Nature, 303: 390). (Produced as described in -396) in a cover glass at a concentration of 3000-5000 cells. O grown in this way
-2A progenitor cells all differentiated into type 2 astrocytes during 4 days of in vitro growth, while cells grown in chemically defined media that were not regulated by endothelial cells were all oligosaccharides. Differentiates into glial cells. Type 2 astrocytes are GFAP
Recognized as + astrocytes and labeled with A2B5 monoclonal antibody.

Examination of media conditioned by endothelial cell lines derived from cerebral cortex of H2ts6 mice indicates that these endothelial cells are indistinguishable in efficacy and capacity from those secreted by non-transgenic endothelial cells. Secretory activity.

Example 7 Colon Epithelial Cells (1) This example demonstrates that the invention can directly immortalize a new category of epithelial cells that is difficult to handle with the application of in vitro gene insertion methods for the generation of immortalized cell lines.

The colon was removed from 14-18 day old H2ts6 mice.
The colon with 0.04% sodium hypochlorite (in PBS)
Washed and sterilized. In one case, the tissue was reduced to 3 mM EDTA +
Crypts were removed from the colon by incubation in 0.05 mM dithiothreitol for 1.5 hours. Organization
Washed with PBS and shaken by hand to separate complete crypts from surrounding tissue. These crypts, which appear as finger-like clusters of cells, are described by Ruff et al. (1983, Nature, 303: 390-396) (Raff et al. (1
983, Nature, 303: 390-396)), plus 2% fetal calf serum + 20 U / ml IFN gamma +
Tumor system LIM1863 (Whitehead et al., 1987, Cancerless, 47: 2683-2689 (Whitehead et al., 1987, Cancer
Res., 47: 2683-2689)) containing Dulbecco's Modified Eagle's Medium containing a 20% conditioned medium (conditioned for 24 hours).
Were grown in monolayer culture on rattail collagen substrate on medium. The crypts settle and the epithelium begins to spread. In the case of non-transgenic crypts, the cells cannot be passaged but can grow on the support layer of bovine vena cava endothelial cells (Whitehead et al., 1991, J. Tissue Cult Methods (Whitehead et al., 1991, J. In contrast, cells from transgenic animals can be grown and passaged without a support layer and established in monolayer culture, in other cases, explant cultures. The colon grew in.
The medium used in all cases is as described above.
In the culture, a new interferon was added a few times a week.

Crypt cultures yielded patches of flat cells with well-defined epithelioid morphology, and explant cultures yielded hybrid cultures containing several morphologically different cell types. Crypt-derived epithelioid cells were labeled with anti-keratin antibodies (LE61 and LP34 listed in Lane, see below), which showed a fibril pattern characteristic of cytoplasmic staining.

Thus, the use of these mice is a means by which colon epithelial cells can be established in culture in a manner not heretofore possible, and from tissues previously not able to be cultured for more than 24-28 hours. I will provide a. The following examples describe the main cell types of interest in explant culture.

Example 8 Enteric Nervous System Glial Cells Colon explant cultures prepared as described in the above examples contained two cell types of particular interest. One of these cells is a small processed cell labeled with an antibody to GFAP, and thus it is recognized as one of the enteric nervous system glial cells. These cells should be easily passaged and easily converted to a cell line. Although there has been considerable interest in glial cells of the enteric nervous system, so far no cell line has been published for this tissue.

The second cell type had a fibroblast-like morphology but was not labeled with anti-GFAP antibodies. However, upon treatment with transforming growth factor-β, these cells are induced to express nestin, an intermediate filament protein that is thought to be specifically expressed by progenitors of the central nervous system (Render, Cell, 19
90, 60: 585-595 (Lendahl, Cell, 1990, 60: 585-59
Five)). Although the normal cellular counterpart of these cells is not known, their induced nestin expression indicates that they may be a novel precursor population.

Example 9 Myoblasts (1) This example demonstrates that cell lines generated by the methods of the present invention retain the ability to undergo normal differentiation in vitro. (2) In addition, this example demonstrates the potential utility of H2ts6 mouse-derived cell lines for undergoing normal differentiation in vivo, and thus the H2ts6 mouse-derived cell lines for cell transplantation.

[0125] Cells isolated from skeletal muscle of the hind legs of newborn mice were
Standard tissue culture conditions for growth of muscle progenitor cells (eg, Morgan et al., 1987, Jay Muscle Les and Cell Motil, 8: 386-), except in the presence of gamma interferon at 0 ° C. 396 (Morgan et al., 1987,
Clonal myoblast cultures were made by direct limiting dilution cloning in J. Muscle Res. And Cell Motil., 8: 386-396). Prior to transplantation of cells into the skeletal muscle mass of non-transgenic mice with known mutations in the dystrophin gene, clone cultures were continuously grown for several weeks and repeatedly passaged. A dystrophin gene mutation implies that this protein exhibits abnormal localization within the multinucleated myotubes formed by fusion and differentiation of muscle progenitor cells. Transplanted cells from transgenic mice could be easily identified by generating skeletal myotubes expressing normal dystrophin. Furthermore, muscle precursor cells from H2ts6 transgenic mice can be fused to polynuclear myotubes in vivo, either by growing cells at high density or by blocking oncogene expression by cell growth under non-permissive conditions. Was.

The cell lines and cell lines of the present invention in which the expression of a differentiation-inhibiting gene is regulated can be obtained by obtaining cell lines and cell lines from any tissue of the body and selecting any cell line or cell line. It differs from the currently feasible cell lines in that it is theoretically possible. Thus, the technology of the present invention was to transfer or infect genetic information into cells in such a way that they could not target specific populations or reliably immortalize rare cells. It is qualitatively different from the conventional technology. This new technology separates rare cells by any conventional means (fluorescence-activated cell sorting, density centrifugation, panning, immunoselection using magnetic beads, selective attachment to defined proteins or carbohydrate substrates, etc.) It can grow under conditions that support the activity of the differentiation-inhibiting gene product, and thus can grow rare cells in large quantities.
Any use of such cells has, for the first time, become a real practical possibility, including (but not limited to) the separation of cellular components or substances from such rare cells.

The immortalized or differentiated cells and immortalized cell lines, differentiated cell lines derived from the animals of this invention have several important uses, particularly including other specific aspects of the invention, such as: Have.

A) An immortalized cell obtained by the above method, or a differentiated cell obtained therefrom, or a cell isolated from the animal of the present invention, which activates the expression of the differentiation inhibitory sequence, Cells of this invention capable of inducing cell differentiation by exposure to an external factor, wherein said exposed cells, or said cells are propagated in vitro and then a non-conditionally immortalizing gene is inserted in vitro As a source of cell-forming substances, any growth or differentiation factor, of cells isolated from
Alternatively use in assay systems related to such substances. (In one exemplary embodiment of this use, the cell-forming substance is an antibody.) B) Immortalized cells obtained by the above method, or differentiated cells obtained therefrom, or isolated from an animal of the present invention. A cell, which activates the expression of the differentiation inhibitory sequence, but is capable of inducing cell differentiation by exposure to an external factor, such exposed cells, or the cells being expanded in vitro, Use of cells isolated from an animal of the invention in which a non-conditional immortalizing gene has been inserted in vitro in the manufacture of a medicament, wherein said medicament is characterized by cell deficiency or cell production factor deficiency. Any of the above, wherein the medicament is a medicament for treating or preventing dysfunction by cell transplantation, or the medicament is a medicament comprising a cell producing factor derived from any of the above cells. Use of the cells in the manufacture of a medicament. C) A particularly important point is the immortalized cells obtained by the above method, or the differentiated cells obtained therefrom, or the cells isolated from the animal of the present invention, wherein the expression of the differentiation inhibitory sequence is activated. However, cells that can be induced to differentiate by exposure to external factors can be induced to differentiate into cells, or cells that have been exposed to such cells, or cells in which the cells have been expanded in vitro, and then a non-conditionally immortalizing gene has been inserted in vitro. The present invention relates to a method for treating or preventing a human or animal body, which comprises administering a cell isolated from an animal or a factor derived from any of the above cells. Specific examples include immortalized cells obtained by the above method, differentiated cells obtained by inactivating the expression of the differentiation inhibitory sequence, and cells isolated from the animal of the present invention. A cell that activates the expression of the differentiation-inhibiting sequence, but is capable of inducing cell differentiation by exposure to an external factor, and that has been exposed to the cells, or a cell isolated from the animal of the present invention, Cells grown in vitro and then into which an unconditional immortalizing gene has been inserted in vitro, under conditions that permit differentiation of the immortalized cells or, in the case of differentiated cells, inhibit the expression of the differentiation inhibitory sequence. There are transplantation methods of transplantation performed on the human or animal body that are transplanted into the body and thereby compensate for the deficiency or dysfunction of existing cells in the body (in an embodiment of such a method, the transplanted cells are An insulin-producing cells or their precursor cells derived from the pancreas,
In this case, the transplantation treatment is for treating or preventing insulin deficiency, or is a glial or progenitor cell,
In this case, the transplantation treatment is for treating or preventing a disease or disorder of the nervous system). D) An immortalized cell obtained by the above method, or a differentiated cell obtained therefrom, or a cell isolated from the animal of the present invention, wherein the expression of the differentiation inhibitory sequence is activated, Can induce cell differentiation by exposure to
The cells so exposed, or the cells isolated from the animal of this invention into which the cells have been grown in vitro and subsequently inserted in vitro with an unconditionally immortalizing gene, in a method of in vitro diagnostics, use.

Cell lines and cell lines are already commonly used as assay systems in the purification of factors that stimulate cell division and differentiation. Cell lines and cell lines can also be used as assay systems for the purification of genes that regulate division and differentiation. Many established oncogenes have been identified by their ability to convert established cell lines to a neoplastic state. Genes that induce cells to differentiate into specific cell types can also be obtained by transfection of genetic material into appropriate recipient cells, as evidenced by recent studies on the identification of genes that regulate myocyte differentiation ( Transfer).
For example, it is conceivable to use an insulin-negative pancreatic cell line as a suitable assay system for identifying factors or genes that induce certain pancreatic cells to make insulin.

Overall, the present invention provides a means that allows the production of a large number of differentiated or progenitor cells. For example, if large numbers of specific types of cells are required for use in diagnostics, transplantation into an individual, or as a means of producing a desired product (eg, a mitogen or differentiation factor), then use known techniques. Appropriate cells can be selected. These cells can then be grown under permissive conditions for a desired period of time. Cells can be examined under permissive conditions that allow at least some differentiation to be seen. In addition, cells can be switched to non-permissive conditions suitable for broader differentiation along the normal pathway. Further, cells can be genetically engineered in tissue culture to express a wild-type differentiation inhibiting sequence,
Thus, large numbers of cells can be grown without continued use of permissive growth conditions (eg, for purification of the desired protein produced by the cells).

It is apparent from the principles of the present invention that conditionally immortalized cells from the transgenic animals of the present case can be introduced into an individual (under non-permissive conditions) and survive in a non-permissive environment there. is there. The introduction of such cells is invaluable for the development of precursor transplantation therapies in which large numbers of progenitor cells are transplanted into affected tissues to replenish the population required for normal function. For example, insulin-producing cells from the pancreas of the transgenic animal of the invention can be surgically transplanted into the pancreas of an animal suffering from insulin deficiency (eg, type I diabetes).

The use of genetically engineered cells to accelerate the regeneration process and restore tissue function is of increasing practical interest. For example, Gage and his colleagues (Science, 1988, 242, 1575) (Gage and colleagues (Scienc
e, 1988, 242, 1575)) describe the injection of fibroblasts genetically engineered to overproduce nerve growth factor into the hippocampal fornix lesion. These transplanted cells have been shown in earlier studies to prevent retrograde axonal degeneration for at least 2 weeks, but more recently to promote cholinergic neuron survival by up to 8 weeks. (Rosenberg et al., 1989, Am Sok Neurosai Abs, No.4
33.2 (Rosenberg, et al, 1989 Am. Soc. Neurosci. Abs. No.
433.2)).

[0133] Many tumors are histologically associated with cells found at the intended stage of tissue development. The animals of the present invention provide a convenient source of cells for the identification of potential precursors of tumor cells. In addition to the strategies already described in connection with the identification of putative glioma progenitor cells, other techniques for gene insertion (e.g., to further manipulate the genome of any cell line isolated from the animal of the invention, such as , Transfection, electroporation, retrovirus-mediated gene insertion) can also be used.
Thus, a sufficient number of specific progenitor or differentiated cell types can be initially grown to allow the use of lower efficiency gene insertion methods, and these further modified cells are replaced with new ones of defined cell types. It can be used to study biological transformation.

The invention also carries a pre-selected mutation,
And a method for providing an animal having cells harboring a sequence encoding a differentiation-inhibiting product that is chromosomally integrated and becomes conditionally active, comprising the steps of providing the aforementioned transgenic animal of the present invention, Crossing a transgenic animal with a parent of a mutant animal exhibiting a preselected mutant phenotype, and obtaining a progeny carrying the mutation and the chromosomally integrated sequence. A method for providing an animal comprising cells carrying a sequence encoding a differentiation-inhibiting product that is chromosomally integrated and has a mutated mutation, and an animal obtained by the method or an animal obtained by the method. Provide offspring.

In addition, the animals of the present invention provide a means to obtain an immortalized cell line that exhibits a preselected mutant phenotype. Therefore,
Another aspect of the invention uses the above-described transgenic animal of the present invention as a parent for crossing and crosses the transgenic animal of the present invention with a parent of a mutant animal exhibiting a preselected mutant phenotype. Thereby producing a progeny mutant animal, wherein said progeny mutant animal exhibits normal cell development and wherein said immortalizable cells exhibiting said mutant phenotype are isolated from said progeny mutant animal. A method of using the transgenic animal of the present invention as a crossing parent for the production of a progeny mutant animal. In such a method of use, it is desirable that both parents be homozygous for each feature exhibited by the progeny produced by the method of use as described above. Therefore, the present invention
It has the additional aspect of providing an immortalized or immortalizable cell from a progeny that exhibits the preselected mutant phenotype and is produced by the method of use as described above.

It will be apparent to those skilled in the art that various modifications and variations can be made to the various embodiments described above without departing from the scope of the inventive concept.

[Brief Description of the Drawings]

FIG. 1. Gene construct H-2K ^b t described in the examples of the present invention.
Schematic diagram of sA58

FIG. 2: Growth analysis showing that control of the biological activity of SV40tsA58 occurs according to the principles of the invention.

FIG. 3 DNA under various conditions for cardiac fibroblasts derived from a mouse according to the invention and having the gene construct of FIG. 1
Diagram showing synthesis

Continuing on the front page (72) Inventor Noble, Mark, David London W. 1 P. 8 Beatty Riding House Street 91 Courtold Building Ludwig Institut For Cancer Researcher (72) Inventor Jatte, Palmjit, Singh London 1-Piece 8 Beatty Riding House Street 91 Court Building Bld. P8 Beatty Riding House Street 91 Courthouse Building Ludwig Institute for Cancer Research Examiner Well Keiko (56) references WO 89/9816 (WO, A1) Dieter Paul et ai l. , Exp. Cell Res. , Vol. 175, pp. 354-362 (1988) See Morello et al. , Oncogene Res. , Vol. 111-124 (1989) Efrat S .; et al. , Proc. Natl. Acad. Sci. USA. , Vol. 85 pp. 9037-9041 (1988) Galiana E. et al. et al. , Journal of Neuroscience Research, vol. 26 pp. 269-277 (1990) Sigmund C.M. D. et al. , J. et al. Biol. Chem. , Vo l. 265 pp. 19916-10022 (1990) Tegtmeyer P. et al. et al. , J. et al. Virol. , Vol. 15, pp. 613-618 (1975) Picard D.C. et al. , Cell, vol. 54, pp. 1073-1080 (1988) Palmiter R. D. et al. , Nature, vol. 316, p. 457-460 (1985) Silers M .; et al. , Nature, vol. 340, pp. 66-68 (1989) (58) Fields investigated (Int. Cl. ⁷ , DB name) A01K 67/027 C12N 5/10 C12N 15/85 BIOSIS / MEDLINE / WPID S (STN)

Claims (19)

(57) [Claims] 1. A transgenic non-human eukaryote having a germ cell and / or a somatic cell into which a sequence has been integrated into a chromosome, wherein the sequence encodes a conditionally active TAgts differentiation inhibitory product. And the sequence is under the control of an activatable promoter, and in vivo, the conditionally active TAgts differentiation inhibitory product enables normal development of the cells in the animal. In culture, given the permissive conditions, the differentiation-inhibiting product, which is TAgts that becomes conditionally active, completes the differentiation of the cells removed from the animal when given permissive conditions during culture. A transgenic non-human eukaryotic animal having germline and / or somatic cells with chromosomally integrated sequences, said expression having a functional level of expression sufficient to prevent chromosomal integration. 2. The animal of claim 1, wherein said promoter is an HLA class I promoter. 3. The animal according to claim 2 wherein the promoter is a HLA H-2K ^b promoter. 4. The animal according to claim 1, wherein the promoter has a plurality of genetic regulatory elements operably linked. 5. The animal according to claim 1, which is a mouse or a rat. 6. carrying a preselected mutation; and
A method for providing an animal comprising a cell having a chromosome-integrated cell having the sequence defined in claim 1, wherein the step of providing the animal according to any one of claims 1 to 5, Crossing an animal according to any one of claims 1 to 5 with a parent of a mutant animal exhibiting a preselected mutant phenotype, and progeny bearing said mutation and said chromosomally integrated sequence. A method for providing an animal comprising cells carrying a preselected mutation and carrying a chromosomally integrated sequence as defined in claim 1 comprising the step of obtaining. 7. An animal obtained by the method of claim 6 or a cell comprising a cell carrying a preselected mutation and carrying a chromosomally integrated sequence as defined in claim 1. progeny. 8. A method for establishing cells in culture, comprising the steps of: removing cells from an animal according to any one of claims 1 to 5 or 7; A method of establishing cells in culture comprising the step of subjecting cells to permissive conditions, wherein said permissive conditions lead to expression of a functional level of said differentiation-inhibiting product to prevent completion of said cell differentiation. 9. The cell of claim 8, wherein said cell is selected from the group consisting of a pancreatic cell, an insulin-producing cell precursor, a glial cell, a glial cell precursor, a muscle cell and a muscle cell precursor.
The method described in. 10. The method according to claim 8, further comprising the step of giving an external factor to the culture to induce differentiation. 11. An isolated cell obtained by the method according to claim 8, 9 or 10. 12. The isolated cell according to claim 11, for treating a human or animal body. 13. The method for producing an expression product of a cell according to claim 11, comprising culturing the cell under conditions suitable for producing the expression product. A method for producing an expression product of 14. A method for producing a transgenic non-human eukaryotic animal, wherein the animal has a construct having a sequence encoding a differentiation inhibitory product that is TAgts that is conditionally active,
The sequence is under the control of an activatable promoter, and in vivo, the conditionally active TAgts differentiation inhibitory product is sufficient to permit normal development of the cells in the animal. Having a low functional level and given permissive conditions in culture, the conditionally active TAgts, a differentiation inhibitory product, is sufficient to prevent the completion of differentiation of cells removed from the animal. A method for producing a transgenic non-human eukaryotic animal, comprising performing chromosomal integration of the sequence into at least a part of the cells of the animal, wherein the method has expression at a functional level. 15. The method of claim 14, wherein the chromosomal integration is performed by microinjection techniques at the embryonic stage of animal development. 16. The method according to claim 14, wherein the promoter is an HLA class I promoter. 17. The method of claim 16 wherein the promoter is a HLA H-2K ^b promoter. 18. The method according to any one of claims 14 to 17, wherein said promoter has a plurality of operably linked genetic regulatory elements. 19. The method according to claim 14, wherein the animal is a mouse or a rat.